Methods of identifying compounds that modulate IL-4 receptor-mediated IgE synthesis utilizing a CLLD8 protein

ABSTRACT

The present provides compounds capable of modulating IL-4 receptor-mediated IgE production, as well as IL-4 induced processes associated therewith, methods and kits for identifying such compounds that utilize a CLLD8 protein as a surrogate analyte and methods of using the compounds in a variety of in vitro, in vitro and ex vivo contexts.

FIELD OF THE INVENTION

[0001] The present invention relates to compounds that modulateprocesses associated with isotype switching of B cells and IgEproduction, methods and kits for identifying such compounds and methodsof using such compounds in a variety of contexts, such as for thetreatment or prevention of diseases associated with or characterized byproduction and/or accumulation of IgE, including anaphylactichypersensitivity or allergic reactions, allergic rhinitis, allergicconjunctivitis, systemic mastocytosis, hyper IgE syndrome, and IgEgammopathies, atopic disorders such as atopic dermatitis, atopic eczemaand atopic asthma, and B-cell lymphoma.

BACKGROUND OF THE INVENTION

[0002] The immune system protects the body against invasion by foreignenvironmental agents such as microorganisms or their products, foods,chemicals, drugs, molds, pollen, animal hair or dander, etc. The abilityof the immune system to protect the body against such foreign invadersmay be innate or acquired.

[0003] The acquired immune response, which stems from exposure to theforeign invader, is extremely complex and involves numerous types ofcells that interact with one another in myriad ways to express the fullrange of immune response. Two of these cell types come from a commonlymphoid precursor cell but differentiate along different developmentallines. One line matures in the thymus (T-cells); the other line maturesin the bone marrow (B-cells). Although T- and B-cells differ in manyfunctional respects, they share one of the important properties of theimmune response: they both exhibit specificity towards a foreign invader(antigen). Thus, the major recognition and reaction functions of theimmune response are contained within the lymph cells.

[0004] A third cell type that participates in the acquired immuneresponse is the class of cells referred to as antigen-presenting cells(APC). Unlike the T- and B-cells, the APC do not haveantigen-specificity. However, they play an important role in processingand presenting the antigen to the T-cells.

[0005] While the T- and B-cells are both involved in acquired immunity,they have different functions. Both T- and B-cells have antigen-specificreceptors on their surfaces that, when bound by the antigen, activatethe cells to release various products. In the case of B-cells, thesurface receptors are immunoglobulins and the products released by theactivated B-cells are immunoglobulins that have the same specificity forthe antigen as the surface receptor immunoglobulins. In the case ofactivated T-cells, the products released are not the same as theirsurface receptor immunoglobulins, but are instead other molecules,called cytokines, that affect other cells and participate in theelimination of the antigen. One such cytokine, released by a class ofT-cells called helper T-cells, is interleukin-4 (IL-4).

[0006] The immunoglobulins produced and released by B-cells must bind toa vast array of foreign invaders (antigens). All immunoglobulins sharecertain common structural features that enable them to: (1) recognizeand bind specifically to a unique structural feature on an antigen(termed an epitope); and (2) perform a common biological function afterbinding the antigen. Basically, each immunoglobulin consists of twoidentical light (L) chains and two identical heavy (H) chains. The Hchains are linked together via disulfide bridges. The portion of theimmunoglobulin that binds the antigen includes the amino-terminalregions of both L and H chains. There are five major classes of Hchains, termed α, δ, ε, γ and μ, providing five different isotypes ofimmunoglobulins: IgA, IgD, IgE, IgG and IgM. Although all five classesof immunoglobulins may possess precisely the same specificity for anantigen, they all have different biological functions.

[0007] While the immune system provides tremendous benefits inprotecting the body against foreign invaders, particularly those thatcause infectious diseases, its effects are sometimes damaging. Forexample, in the process of eliminating an invading foreign substancesome tissue damage may occur, typically as a result of the accumulationof immunoglobulins with non-specific effects. Such damage is generallytemporary, ceasing once the foreign invader has been eliminated.However, there are instances, such as in the case of hypersensitivity orallergic reactions, where the immune response directed against eveninnocuous agents such as inhaled pollen, inhaled mold spores, insectbite products, medications and even foods, is so powerful that itresults in severe pathological consequences or symptoms.

[0008] Such hypersensitivity or allergic reactions are divided into fourclasses, designated types I-IV. The symptoms of the type I allergicreactions, called anaphylactic reactions or anaphylaxis, include thecommon symptoms associated with mild allergies, such as runny nose,watery eyes, etc., as well as the more dangerous, and often fatal,symptoms of difficulty in breathing (asthma), asphyxiation (typicallydue to constriction of smooth muscle around the bronchi in the lungs)and a sharp drop in blood pressure. Also included within the class oftype I allergic reactions are atopic reactions, including atopicdermatitis, atopic eczema and atopic asthma.

[0009] Even when not lethal, such anaphylactic allergic reactionsproduce symptoms that interfere with the enjoyment of normal life. Oneneed only witness the inability of an allergy sufferer to mow the lawnor hike through the woods to understand the disruptive force even mildallergies have on everyday life. Thus, while the immune system is quitebeneficial, it would be desirable to be able to interrupt its responseto invading foreign agents that pose no risk or threat to the body.

[0010] IgE immunoglobulins are crucial immune mediators of suchanaphylactic hypersensitivity and allergic reactions, and have beenshown to be responsible for the induction and maintenance ofanaphylactic allergic symptoms. For example, anti-IgE antibodies havebeen shown to interfere with IgE function and alleviate allergicsymptoms (Jardieu, 1995, Curr. Op. Immunol. 7:779-782; Shields et al.,1995, Int. Arch. Allergy Immunol. 107:308-312). Thus, release and/oraccumulation of IgE immunoglobulins are believed to play a crucial rolein the anaphylactic allergic response to innocuous foreign invaders.Other diseases associated with or mediated by IgE production and/oraccumulation include, but are not limited to, allergic rhinitis,allergic conjunctivitis, systemic mastocytosis, hyper IgE syndrome, IgEgammopathies and B-cell lymphoma.

[0011] Although IgEs are produced and released by B-cells, the cellsmust be activated to do so (B-cells initially-produce only IgD and IgM).The isotype switching of B-cells to produce IgE is a complex processthat involves the replacement of certain immunoglobulin constant (C)regions with other C regions that have biologically distinct effectorfunctions, without altering the specificity of the immunoglobulin. ForIgE switching, a deletional rearrangement of the IgH chain gene locusoccurs, which results in the joining of the switch region of the μ gene,Sμ, with the corresponding region of the ε gene, Sε.

[0012] This IgE switching is induced in part by IL-4 (or IL-13) producedby T-cells. The IL-4 induction initiates transcription through the Sεregion, resulting in the synthesis of germline (or “sterile”) εtranscripts (that is, transcripts of the unrearranged Cε H genes) thatlead to the production of IgE instead of IgM.

[0013] IL-4 induced germline ε transcription and consequent synthesis ofIgE is inhibited by interferon gamma (IFN-γ), interferon alpha (IFN-α)and tumor growth factor beta (TGF-β). In addition to the IL-4 signal, asecond signal, also normally delivered by T-cells, is required forswitch recombination leading to the production of IgE. This secondT-cell signal may be replaced by monoclonal antibodies to CD40,infection by Epstein-Barr virus or hydrocortisone.

[0014] Generally, traditional treatments for diseases mediated by IgEproduction and/or accumulation regulate the immune system followingsynthesis of IgE. For example, traditional therapies for the treatmentof allergies include anti-histamines designed to modulate theIgE-mediated response resulting in mast cell degranulation. Drugs arealso known that generally downregulate IgE production or that inhibitswitching of, but not induction of, germline ε transcription (see, e.g.,Loh et al., 1996, J. Allerg. Clin. Immunol. 97(5):1141).

[0015] Although these treatments are often effective, treatments thatact to reduce or eliminate IgE production altogether would be desirable.By reducing or eliminating IgE production, the hypersensitivity orallergic response may be reduced or eliminated altogether. Accordingly,the availability of compounds that are upstream modulators of IgEproduction, such as compounds capable of modulating, and in particularinhibiting, IL-4 receptor-mediated germline ε transcription, would behighly desirable.

[0016] The ability to screen for compounds capable of modulating IgEproduction, and in particular compounds that modulate IL-4 (or IL-13)induced germline ε transcription typically involves screening candidatecompounds in complex cell-based functional assays, such as thefunctional assays described in U.S. Pat. No. 5,958,707. These assaystypically involve contacting a cell comprising a reporter gene operablylinked to a promoter responsive to or inducible by IL-4 with a candidatecompound of interest in the presence of IL-4 and assessing the amount ofreporter gene product produced. The reporter gene is typically a genethat encodes a protein that produces an observable signal, such as afluorescent protein. The IL-4 inducible promoter may be a germline εpromoter. Compounds that antagonize (inhibit) IL-4 induced transcriptionwill yield reduced amounts of reporter gene product as compared tocontrol cells contacted with IL-4 alone. Compounds that agonize IL-4induced transcription will yield increased amounts of reporter geneproduct as compared to control cells contacted with IL-4 alone.Particularly useful functional assays for screening compounds for theability to modulate IL-4 inducible germline ε transcription aredescribed in U.S. Pat. No. 5,958,707, WO 99/58663 and WO 01/34806.

[0017] Although such functional screening assays are quite powerful andeffective, simpler assays that could be performed in cell-free systemsand/or that do not require a functional component, such as simplebinding assays with isolated proteins known to be involved in the IL-4signaling cascade responsible for the production of germline εtranscripts, and hence the production of IgE, would be beneficial.

SUMMARY OF THE INVENTION

[0018] These and other objects are furnished by the present invention,which in one aspect provides compounds, referred to herein as CL02A3compounds, which are capable of modulating, and in particularinhibiting, the IL-4 receptor-mediated signaling cascade involved inB-cell isotype switching to, and consequent production of, IgE. TheCL02A3 compounds of the invention are generally 8 to 30 amino acidresidue peptides or peptide analogs, or pharmaceutically-acceptablesalts thereof, characterized by structural formula (I):

Z¹-X¹˜X²˜X⁴˜X⁵˜X⁶˜X⁷˜X⁸X⁹˜X¹⁰˜X¹¹X¹²˜X¹³˜Z¹⁴˜X¹⁵˜X¹⁶˜X¹⁷˜X¹⁹˜X¹⁹˜X²⁰-Z²

[0019] wherein:

[0020] X¹ is a small aliphatic residue;

[0021] X² is a non-polar residue;

[0022] X³ is an aromatic residue, a basic residue or a Ala residue;

[0023] X⁴ is a Gly or Ala residue;

[0024] X⁵ is an aromatic residue, a basic residue or a Ala residue;

[0025] X⁶ is an aromatic residue a basic residue or a Ala residue;

[0026] X⁷ is an aliphatic residue;

[0027] X⁸ is an aliphatic residue or an aromatic residue;

[0028] X⁹ is a conformationally-constrained residue or a Ala residue;

[0029] X¹⁰ is an aromatic residue or a Ala residue;

[0030] X¹¹ is a Gly or Ala residue;

[0031] X¹² is a non-polar residue;

[0032] X¹³ is an acidic residue or a Ala residue;

[0033] X¹⁴ is a polar residue or a Ala residue;

[0034] X¹⁵ is a Gly or Ala residue;

[0035] X¹⁶ is a cysteine-like residue or a Ala residue;

[0036] X¹⁷ is a hydroxyl-containing residue or a Ala residue;

[0037] X¹⁸ is a hydrophobic residue;

[0038] X¹⁹ is an aliphatic residue;

[0039] X²⁰ is an aliphatic residue;

[0040] Z¹ is RRN—, RC(O)NR—, RS(O)₂NR— or an amino-terminal blockinggroup;

[0041] Z² is —C(O)OR, —C(O)O—, —C(O)NRR or a carboxyl-terminal blockinggroup;

[0042] each R is independently selected from the group consisting ofhydrogen, alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl,substituted arylalkyl, heteroaryl, substituted heteroaryl,heteroarylalkyl and substituted heteroarylalkyl;

[0043] each “˜” independently represents an amide, a substituted amideor an isostere of an amide;

[0044] each “-” represents a bond, or a 1 to 10 residue peptide orpeptide analog; and

[0045] wherein one or more of X¹, X², X¹⁹, or X²⁰ may be absent.

[0046] The CL02A3 compounds of the invention preferably have between 10and 25 residues; more preferably, the CL02A3 compounds of the inventionhave between 12 and 23 residues. In particular, the more preferredembodiments have 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23residues. Particular CL02A3 compounds of the invention include:CL02A3wt, (AMHGHHGWPWGMEQGCTPLG SEQ ID NO:1) CL02A3LG,(AMHGHHGWPWGMEQGCTPAA SEQ ID NO:2) CL02A3GW, (AMHGHHAAPWGMEQGCTPLG SEQID NO:3) CL02A3TP, (AMHGHHGWPWGMEQGCAALG SEQ ID NO:4) CL02A3EQ,(AMHGHHGWPWGMAAGCTPLG SEQ ID NO:5) CL02A3HG, (AMAAHHGWPWGMEQGCTPLG SEQID NO:6) CL02A3GC, (AMHGHHGWPWGMEQAATPLG SEQ ID NO:7) CL02A3HH,(AMHGAAGWPWGMEQGCTPLG SEQ ID NO:8) and CL02A3PW. (AMHGHHGWAAGMEQGCTPLGSEQ ID NO:9)

[0047] The CL02A3 compounds of the invention inhibit IL-4 (or IL-13)induced germline ε transcription in cellular assays. As a consequence ofthis activity, the CL02A3 compounds of the invention can be used tomodulate the IL-4 receptor-mediated signaling cascade involved inisotype switching to, and consequent production of, IgE. In a specificembodiment, the CL02A3 compounds may be used to inhibit IL-4 (or IL-13)induced IgE production as a therapeutic approach towards the treatmentor prevention of diseases associated with, characterized by or caused byIgE production and/or accumulation, such as anaphylactichypersensitivity or allergic reactions and/or symptoms associated withsuch reactions, allergic rhinitis, allergic conjunctivitis, systemicmastocytosis, hyper IgE syndrome, and IgE gammopathies, atopic disorderssuch as atopic dermatitis, atopic eczema and atopic asthma, and B-celllymphoma.

[0048] The CL02A3 compound of the invention can also be used in assaysto identify other compounds capable of effecting the above activities,as will be described in more detail, below.

[0049] In addition to their ability to inhibit IL-4 (or IL-13) induciblegermline ε transcription, and hence IL-4 receptor-mediated IgEproduction, the CL02A3 compound of the invention also bind CLLD8proteins. In particular, three CL02A3 compounds of the invention,peptide CL02A3 wt (AMHGHHGWPWGMEQGCTPLG; SEQ ID NO:1), peptide CL02A3LG(AMHGHHGWPWGMEQGCTPAA; SEQ ID NO:2) and peptide CL02A3GW(AMHGHHAAPWGMEQGCTPLG; SEQ ID NO:3), were found to bind human CLLD8protein (hCLLD8) in a yeast two hybrid interaction assay. Quitesignificantly, the ability of these CL02A3 compounds to bind the hCLLD8protein in this assay correlates with their observed ability to inhibitIL-4 (or IL-13) induced germline ε transcription. These observationsprovide the first evidence directly linking CLLD8 protein to the IL-4receptor-mediated signaling cascade responsible for isotype switchingto, and consequent production of, IgE, and in particular as being aneffector of germline ε transcription. Hence, these observations providethe first evidence directly linking CLLD8 protein to the upstreamregulation of isotype switching and/or IgE production.

[0050] This significant discovery enables the ability to use a CLLD8protein as a “surrogate” analyte in screening assays to identifycompounds that modulate or regulate the IL-4 receptor-mediated signalingcascade leading to the production of IgE. Since induction of the εpromoter in response to IL-4 (or IL-13) is the first recognizable stepnecessary for isotype switching and consequent production of IgE,inhibition of IL-4 (or IL-13) induced transcription of the ε promotershould prevent B-cells from switching to and/or producing IgE. Thissignificant discovery therefore permits the ability to use a CLLD8protein as a surrogate analyte in simple binding assays to identifycompounds having a variety of in vitro, in vivo and ex vivo therapeuticuses.

[0051] Accordingly, in another aspect, the invention provides methods ofidentifying compounds that modulate, and in particular inhibit, the IL-4receptor-mediated signaling cascade leading to the production of IgE.The methods generally comprise determining whether a candidate compoundof interest binds a CLLD8 protein, wherein the ability to bind the CLLD8protein identifies the compound as being a modulator of the IL-4receptor-mediated signaling cascade leading to the production of IgE. Inone embodiment of the method, it is determined whether the candidatecompound competes for binding to the CLLD8 protein with a CL02A3compound of the invention, such as peptide CL02A3 wt, peptide CL02A3LGor peptide CL02A3GW. In a specific embodiment of the method, compoundsthat inhibit IL-4 (or IL-13) induced germline ε transcription areidentified. In a further embodiment, the methods comprise determiningwhether a candidate compound of interest is an inhibitor of CLLD8. Thedetermination of the candidate compound as a CLLD8 inhibitor can be madein addition to, or as an alternative to, determination of the ability ofthe candidate compound to bind to a CL02A3 compound.

[0052] In yet another aspect, the invention provides methods ofidentifying compounds that inhibit isotype switching of B-cells toproduce IgE and/or IgE production. The methods generally comprisedetermining whether a candidate compound of interest binds a CLLD8protein, wherein the ability to bind the CLLD8 protein identifies thecompound as being an inhibitor of isotype switching and/or IgEproduction. In one embodiment of the method, it is determined whetherthe candidate compound competes for binding to the CLLD8 protein with aCL02A3 compound of the invention, such as peptide CL02A3 wt, peptideCL02A3LG or peptide CL02A3GW. In a specific embodiment of the method,compounds that inhibit IgE production are identified. In anotherspecific embodiment, compounds that inhibit IL-4 receptor-mediated IgEproduction are identified. In still another specific embodiment,compounds that inhibit CLLD8 protein-mediated IgE production areidentified.

[0053] In still another aspect, the invention provides methods ofidentifying compounds that modulate, and in particular inhibit ordownregulate, processes mediated by or associated with IL-4receptor-mediated B-cell isotype switching and/or IgE production and/oraccumulation. Such processes include, but are not limited to,anaphylactic hypersensitivity or allergic reactions and/or symptomsassociated with such reactions, allergic rhinitis, allergicconjunctivitis, systemic mastocytosis, hyper IgE syndrome, and IgEgammopathies, atopic disorders such as atopic dermatitis, atopic eczemaand atopic asthma, and B-cell lymphoma. The methods generally involvedetermining whether a candidate compound of interest binds a CLLD8protein, where the ability to bind the CLLD8 protein identifies thecompound as being a modulator of a process mediated by or associatedwith IL-4 receptor-mediated, isotype switching and/or IgE productionand/or accumulation. In one embodiment of the method, it is determinedwhether the candidate compound competes for binding the CLLD8 proteinwith a CL02A3 compound of the invention, such as peptide CL02A3 wt,peptide CL02A3LG or peptide CL02A3GW.

[0054] In yet another aspect, the invention provides methods ofidentifying compounds useful for treating disorders associated with, ormediated or caused by, IgE production and/or accumulation. The methodsgenerally comprise determining whether a candidate compound of interestbinds a CLLD8 protein, wherein the ability to bind the CLLD8 proteinidentifies the compound as being useful for treating disordersassociated with, or mediated or caused by, IgE production and/oraccumulation. In one embodiment of the method, it is determined whetherthe candidate compound competes for binding the CLLD8 protein with aCL02A3 compound of the invention, such as peptide CL02A3 wt, peptideCL02A3LG or peptide CL02A3GW. Diseases associated with, or mediated orcaused by, IgE production and/or accumulation for which therapeuticcompounds may be identified according to the methods include, but arenot limited to, anaphylactic hypersensitivity or allergic reactionsand/or symptoms associated with such reactions, allergic rhinitis,allergic conjunctivitis, systemic mastocytosis, hyper IgE syndrome, andIgE gammopathies, atopic disorders such as atopic dermatitis, atopiceczema and atopic asthma, and B-cell lymphoma.

[0055] In another aspect, the invention provides compounds identified bythe various screening methods of the invention.

[0056] In still another aspect, the invention provides pharmaceuticalcompositions. The compositions generally comprise a CL02A3 compound ofthe invention, a compound that competes for binding a CLLD8 protein witha CL02A3 compound of the invention or a compound identified by thescreening methods of the invention and a pharmaceutically-acceptablecarrier, excipient or diluent.

[0057] In yet another aspect, the invention provides methods ofmodulating, and in particular inhibiting or downregulating, the IL-4receptor-mediated signaling cascade involved in B-cell isotype switchingto produce, and/or consequent production of, IgE, or processes involvedin this signal transduction cascade, such as IL-4 (or IL-13) inducedgermline ε transcription. The method generally involves administering toa cell a compound that binds a CLLD8 protein in an amount effective tomodulate this IL-4 receptor-mediated signaling cascade. In oneembodiment of the method, the compound inhibits IL-4 (or IL-13) inducedgermline ε transcription. In another specific embodiment, the compoundinhibits CLLD8 protein-mediated germline ε transcription. The method maybe practiced in vitro, in vivo or ex vivo. In one embodiment, the cellis administered a CL02A3 compound of the invention, such as peptideCL02A3 wt, peptide CL02A3LG, peptide CL02A3GW, peptide CL02A3TP, peptideCL02A3EQ, peptide CL02A3HG, peptide CL02A3GC, peptide CL02A3HH, peptideCL02A3PW, or a compound that competes for binding to a CLLD8 proteinwith an active CL02A3 compound of the invention.

[0058] In yet another aspect, the invention provides methods ofmodulating, and in particular inhibiting or downregulating, isotypeswitching to IgE and/or IgE production. The method generally involvesadministering to a cell an amount of a compound that binds a CLLD8protein effective to modulate isotype switching to IgE and/or IgEproduction. The method may be practiced in vitro, in vivo or ex vivo. Inone embodiment, the cell is administered a CL02A3 compound of theinvention, such as peptide CL02A3 wt, peptide CL02A3LG, peptideCL02A3GW, peptide CL02A3TP, peptide CL02A3EQ, peptide CL02A3HG, peptideCL02A3GC, peptide CL02A3HH, peptide CL02A3PW, or a compound thatcompetes for binding to a CLLD8 protein with an active CL02A3 compoundof the invention.

[0059] In still another aspect, the invention provides methods oftreating or preventing diseases associated with, or mediated or causedby, IgE production and/or accumulation. The method generally comprisesadministering to an animal suffering from such a disease an amount of acompound that binds a CLLD8 protein effective to treat or prevent thedisease and/or one or more of its symptoms. In one embodiment, thecompound administered is a CL02A3 compound of the invention, such aspeptide CL02A3 wt, peptide CL02A3LG, peptide CL02A3GW, peptide CL02A3TP,peptide CL02A3EQ, peptide CL02A3HG, peptide CL02A3GC, peptide CL02A3HH,or peptide CL02A3PW. In another embodiment, the compound administered isa compound that competes for binding to a CLLD8 protein with an activeCL02A3 compound of the invention. Diseases associated with, or mediatedor caused by, IgE production and/or accumulation that may be treated orprevented according to the methods of the invention include, but are notlimited to anaphylactic hypersensitivity or allergic reactions and/orsymptoms associated with such reactions (including food and drugallergies), allergic rhinitis, allergic conjunctivitis, systemicmastocytosis, hyper IgE syndrome, and IgE gammopathies, atopic disorderssuch as atopic dermatitis, atopic eczema and atopic asthma, and B-celllymphoma. The method may be practiced therapeutically to treat thedisease once the onset of the disease and/or its associated symptomshave already occurred, or prophylactically to prevent the onset of thedisease and/or its associated symptoms. The methods may be practiced inveterinary contexts or in the treatment of humans.

[0060] In a final aspect, the invention provides kits for carrying outthe various methods of the invention. In one embodiment, the kitcomprises a CLLD8 protein and a CL02A3 compound of the invention or acompound that competes for binding the CLLD8 protein with a CL02A3compound of the invention. The kit may further include additionalcomponents for carrying out the methods of the invention, such as, byway of example and not limitation, buffers, labels and/or labelingreagents and/or instructions teaching methods of using the kits.

BRIEF DESCRIPTION OF THE FIGURES

[0061]FIG. 1 illustrates the nucleotide sequence of a 603 bp fragment ofthe human germline ε promoter (SEQ ID NO:17);

[0062]FIG. 2A provides a cartoon illustrating the diphtheria toxin (DT)selection of reporter cell line A5T4;

[0063]FIG. 2B provides a cartoon illustrating thetetracycline/doxycycline controlled peptide expression system ofreporter cell line A5T4;

[0064]FIG. 3 provides a cartoon illustrating the enrichment andscreening procedure used to identify certain CL02A3 compounds of theinvention;

[0065]FIG. 4 provides DNA transfer data for peptide CL02A3 wt;

[0066]FIG. 5A provides a cartoon outlining a yeast two hybrid (YTH)screening assay used to identify potential binding partners or targetsfor peptide CL02A3 wt;

[0067]FIG. 5B provides a cartoon illustrating a yeast two hybrid (YTH)assay used to identify hCLLD8 as a binding partner or target for peptideCL02A3 wt;

[0068]FIG. 6 provides a cartoon summarizing strategies for reconfirmingpotential targets identified in the YTH assay depicted in FIG. 5A;

[0069]FIG. 7 provides interaction graphic profiles for peptide CL02A3 wtand mutants derived therefrom with the putative target cloneCL02A3-NB739. The bar graphs indicate normalized β-galactosidaseactivity;

[0070]FIG. 8 provides selection criteria for the interaction profilingmethod used to confirm human CLLD8 as a binding partner for peptideCL02A3 wt;

[0071]FIG. 9 provides weighted graphic interaction/functional profilesfor peptide CL02A3 wt and mutants derived therefrom for 5 putativetarget clones;

[0072]FIG. 10 illustrates the amino acid sequence of human CLLD8 (SEQ IDNO: 18).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0073] Abbreviations

[0074] The abbreviations used for the genetically encoded amino acidsare conventional and are as follows: Amino Acid Three-LetterAbbreviation One-Letter Abbreviation Alanine Ala A Arginine Arg RAsparagine Asn N Aspartate Asp D Cysteine Cys C Glutamate Glu EGlutamine Gln Q Glycine Gly G Histidine His H Isoleucine Ile I LeucineLeu L Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro PSerine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine ValV

[0075] When the three-letter abbreviations are used, unless specificallypreceded by an “L” or a “D,” the amino acid may be in either the L- orD-configuration about α-carbon (C_(α)). For example, whereas “Ala”designates alanine without specifying the configuration about theα-carbon, “D-Ala” and “L-Ala” designate D-alanine and L-alanine,respectively. When the one-letter abbreviations are used, upper caseletters designate amino acids in the L-configuration about the α-carbonand lower case letters designate amino acids in the D-configurationabout the α-carbon. For example, “A” designates L-alanine and “a”designates D-alanine. When polypeptide sequences are presented as astring of one-letter or three-letter abbreviations (or mixturesthereof), the sequences are presented in the N->C direction inaccordance with common convention.

[0076] The abbreviations used for the genetically encoding nucleosidesare conventional and are as follows: adenosine (A); guanosine (G);cytidine (C); thymidine (T); and uridine (U). Unless specificallydelineated, the abbreviated nucleotides may be either ribonucleosides or2′-deoxyribonucleosides. The nucleosides may be specified as beingeither ribonucleosides or 2′-deoxyribonucleosides on an individual basisor on an aggregate basis. When specified on an individual basis, theone-letter abbreviation is preceded by either a “d” or an “r,” where “d”indicates the nucleoside is a 2′-deoxyribonucleoside and “r” indicatesthe nucleoside is a ribonucleoside. For example, “dA” designates2′-deoxyriboadenosine and “rA” designates riboadenosine. When specifiedon an aggregate basis, the particular nucleic acid or polynucleotide isidentified as being either an RNA molecule or a DNA molecule.Nucleotides are abbreviated by adding a “p” to represent each phosphate,as well as whether the phosphates are attached to the 3′-position or the5′-position of the sugar. Thus, 5′-nucleotides are abbreviated as “pN”and 3′-nucleotides are abbreviated as “Np,” where “N” represents A, G,C, T or U. When nucleic acid sequences are presented as a string ofone-letter abbreviations, the sequences are presented in the 5′->3′direction in accordance with common convention, and the phosphates arenot indicated.

[0077] Definitions

[0078] As used throughout the instant application, the following termsshall have the following meanings:

[0079] “Promoter” or “Promoter Sequence” refers to a DNA regulatoryregion capable of initiating transcription of a downstream (3′direction) coding sequence. A promoter typically includes atranscription initiation site (conveniently defined, for example, bymapping with nuclease SI) and protein binding domains responsible forbinding proteins that initiate transcription.

[0080] “TGF-β Inducible Promoter” refers to a promoter that initiatestranscription when a cell comprising a nucleic acid molecule includingsuch a promoter is exposed to, or contacted with, TGF-β. While notintending to be bound by any particular theory of operation, it isbelieved that contacting a cell comprising such a promoter with TGF-βcauses the activation of a DNA-binding protein that then binds the TGF-βinducible promoter and induces transcription of coding sequencesdownstream of the promoter

[0081] An “α promoter” or a “germline apromoter” is a TGF-β induciblepromoter that, when induced in a B-cell, leads to the production of IgAimmunoglobulins. Such germline α promoters are well-known in the art.Such promoters may be endogenous to a cell, or alternatively, they maybe engineered or exogenously supplied.

[0082] “IL-4 inducible promoter” refers to a promoter that initiatestranscription when a cell comprising a nucleic acid molecule includingsuch a promoter is exposed to, or contacted with, IL-4 or IL-13. Whilenot intending to be bound by any particular theory of operation, it isbelieved that contacting a cell comprising such a promoter with IL-4 (orIL-13) causes the activation of a DNA-binding protein that then bindsthe IL-4 inducible promoter and induces transcription of codingsequences downstream of the promoter.

[0083] An “ε promoter” or a “germline ε promoter” is an IL-4 induciblepromoter that, when induced in a B-cell, leads to the production of IgEimmunoglobulins. Such IL-4 inducible germline ε promoters are well-knownin the art. Such promoters may be endogenous to a cell or,alternatively, they may be engineered or supplied exogenously. Aspecific example of a germline ε promoter is the 600 bp IL-4 induciblefragment of the human ε promoter depicted in FIG. 1 (SEQ ID NO: 17).

[0084] A compound that “modulates an IL-4 inducible germ line εpromoter” or that “modulates IL-4 induced germline ε transcription” hasthe ability to change or alter expression downstream of the germline εpromoter induced by IL-4 (or IL-13). The change in IL-4 induceddownstream expression may occur at the mRNA (transcriptional) level orat the protein (translational) level. Hence, the change in IL-4 induceddownstream expression may be monitored at the RNA level, for example byquantifying induced downstream transcription products, or at the proteinlevel, for example by quantifying the amount or activity of induceddownstream translation products. The compound may act to modulate theIL-4 inducible germline ε promoter via any mechanism of action. Forexample, the compound may act to modulate the IL-4 inducible germline εpromoter by interacting with or binding a DNA binding protein involvedin the IL-4 induced transcription, or by interacting with or binding theIL-4 inducible germline E promoterper se, or by interacting with orbinding to a molecule that functions in the signalling cascade triggeredby IL-4.

[0085] A compound that “modulates IL-4 receptor-mediated IgE productionand/or accumulation” has the ability to change or alter the amount ofIgE produced and/or accumulated by a B-cell activated via the IL-4receptor with IL-4 (or IL-13 or other IL-4 receptor ligand) and, in somecases, a second signal known to cause, in combination with IL-4 (orIL-13), isotype switching of B-cells to produce IgE. Such second signalmay be, for example, anti-CD40 monoclonal antibodies (anti-CD40 mAbs),infection by Epstein-Barr virus or hydrocortisone. The compound may actto modulate IL-4 receptor-mediated IgE production and/or accumulationvia any mechanism of action. For example, the compound may act tomodulate IL-4 induced germline E transcription, and hence isotypeswitching, or the compound may act to modulate IgE production and/oraccumulation in an already switched cell. An “IL-4 induced” activityincludes an activity (e.g., production of IgE, transcription of germlineε promoter, isotype switching of B-cells, etc.) that is induced as aresult of the binding of IL-4, IL-13 or other IL-4 receptor ligand tothe IL-4 receptor.

[0086] “Identifying” in the context of screening assays meansdetermining whether a candidate compound unknown to possess a particularproperty of interest possesses the property of interest, as well asconfirming that a compound thought or known to possess a particularproperty of interest possesses the property of interest.

[0087] Compounds that “competefor binding with a CL02A3 compound”compete for binding to a CLLD8 protein (defined in a later section) withan active CL02A3 compound of invention (described in more detail in alater section), such as peptide CL02A3 wt, peptide CL02A3GW or peptideCL02A3LG, or with another compound that competes for binding to a CLLD8protein with an active CL02A3 compound of the invention. For example, ifcompound 1 competes for binding to a CLLD8 protein with an active CL02A3compound and a candidate compound competes for binding to the CLLD8protein with compound 1, then for purposes of the present invention, thecandidate compound competes for binding with a CL02A3 compound. Wherecompetition with a specific category of compound is intended, themodifiers “directly” and “indirectly” are used, where “directly” refersto competition with the stated compound and “indirectly” refers tocompetition with another compound that itself competes with the statedcompound.

[0088] “Alkyl” by itself or as part of another substituent refers to asaturated or unsaturated, branched, straight-chain or cyclic monovalenthydrocarbon group having the stated number of carbon atoms (i.e., C₁-C₆means from one to six carbon atoms) derived by the removal of onehydrogen atom from a single carbon atom of a parent alkane, alkene oralkyne. Typical alkyl groups include, but are not limited to, methyl;ethyls such as ethanyl, ethenyl, ethynyl; propyls such as propan-1-yl,propan-2-yl, cyclopropan-1-yl, prop-1-en-1-yl, prop-1-en-2-yl,prop-2-en-1-yl (allyl), cycloprop-1-en-1-yl; cycloprop-2-en-1-yl,prop-1-yn-1-yl, prop-2-yn-1-yl, etc.; butyls such as butan-1-yl,butan-2-yl, 2-methyl-propan-1-yl, 2-methyl-propan-2-yl, cyclobutan-1-yl,but-1-en-1-yl, but-1-en-2-yl, 2-methyl-prop-1-en-1-yl, but-2-en-1-yl,but-2-en-2-yl, buta-1,3-dien-1-yl, buta-1,3-dien-2-yl,cyclobut-1-en-1-yl, cyclobut-1-en-3-yl, cyclobuta-1,3-dien-1-yl,but-1-yn-1-yl, but-1-yn-3-yl, but-3-yn-1-yl, etc.; and the like. Theterrn “alkyl” is specifically intended to include groups having anydegree or level of saturation, i.e., groups having exclusively singlecarbon-carbon bonds, groups having one or more double carbon-carbonbonds, groups having one or more triple carbon-carbon bonds and groupshaving mixtures of single, double and triple carbon-carbon bonds. Wherea specific level of saturation is intended, the expressions “alkanyl,”“alkenyl,” and “alkynyl” are used. The expression “lower alkyl” refersto alkyl groups composed of from 1 to 6 carbon atoms.

[0089] “Alkanyl” by itself or as part of another substituent refers to asaturated branched, straight-chain or cyclic alkyl group. Typicalalkanyl groups include, but are not limited to, methanyl; ethanyl;propanyls such as propan-1-yl, propan-2-yl (isopropyl),cyclopropan-1-yl, etc.; butyanyls such as butan-1-yl, butan-2-yl(sec-butyl), 2-methyl-propan-1-yl (isobutyl), 2-methyl-propan-2-yl(t-butyl), cyclobutan-1-yl, etc.; and the like.

[0090] “Alkenyl” by itself or as part of another substituent refers toan unsaturated branched, straight-chain or cyclic alkyl group having atleast one carbon-carbon double bond derived by the removal of onehydrogen atom from a single carbon atom of a parent alkene. The groupmay be in either the cis or trans conformation about the double bond(s).Typical alkenyl groups include, but are not limited to, ethenyl;propenyls such as prop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl(allyl), prop-2-en-2-yl, cycloprop-1-en-1-yl; cycloprop-2-en-1-yl;butenyls such as but-1-en-1-yl, but-1-en-2-yl, 2-methyl-prop-1-en-1-yl,but-2-en-1-yl, but-2-en-1-yl, but-2-en-2-yl, buta-1,3-dien-1-yl,buta-1,3-dien-2-yl, cyclobut-1-en-1-yl, cyclobut-1-en-3-yl,cyclobuta-1,3-dien-1-yl, etc.; and the like.

[0091] “Alkynyl” by itself or as part of another substituent refers toan unsaturated branched, straight-chain or cyclic alkyl group having atleast one carbon-carbon triple bond derived by the removal of onehydrogen atom from a single carbon atom of a parent alkyne. Typicalalkynyl groups include, but are not limited to, ethynyl; propynyls suchas prop-1-yn-1-yl, prop-2-yn-1-yl, etc.; butynyls such as but-1-yn-1-yl,but-1-yn-3-yl, but-3-yn-1-yl, etc.; and the like.

[0092] “Parent Aromatic Ring System” refers to an unsaturated cyclic orpolycyclic ring system having a conjugated π electron system.Specifically included within the definition of “parent aromatic ringsystem” are fused ring systems in which one or more of the rings arearomatic and one or more of the rings are saturated or unsaturated, suchas, for example, fluorene, indane, indene, phenalene, etc. Typicalparent aromatic ring systems include, but are not limited to,aceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene,benzene, chrysene, coronene, fluoranthene, fluorene, hexacene,hexaphene, hexalene, as-indacene, s-indacene, indane, indene,naphthalene, octacene, octaphene, octalene, ovalene, penta-2,4-diene,pentacene, pentalene, pentaphene, perylene, phenalene, phenanthrene,picene, pleiadene, pyrene, pyranthrene, rubicene, triphenylene,trinaphthalene, and the like.

[0093] “Aryl” by itself or as part of another substituent refers to amonovalent aromatic hydrocarbon group having the stated number of carbonring atoms (i.e., C₅-C₁₄ means from 5 to 14 carbon ring atoms) derivedby the removal of one hydrogen atom from a single carbon atom of aparent aromatic ring system. Typical aryl groups include, but are notlimited to, groups derived from aceanthrylene, acenaphthylene,acephenanthrylene, anthracene, azulene, benzene, chrysene, coronene,fluoranthene, fluorene, hexacene, hexaphene, hexalene, as-indacene,s-indacene, indane, indene, naphthalene, octacene, octaphene, octalene,ovalene, penta-2,4-diene, pentacene, pentalene, pentaphene, perylene,phenalene, phenanthrene, picene, pleiadene, pyrene, pyranthrene,rubicene, triphenylene, trinaphthalene, and the like. In preferredembodiments, the aryl group is (C₅-C₁₄) aryl, with (C₅-C₁₀) being evenmore preferred. Particularly preferred aryls are cyclopentadienyl,phenyl and naphthyl.

[0094] “Arylalkyl” by itself or as part of another substituent refers toan acyclic alkyl group in which one of the hydrogen atoms bonded to acarbon atom, typically a terminal or sp³ carbon atom, is replaced withan aryl group. Typical arylalkyl groups include, but are not limited to,benzyl, 2-phenylethan-1-yl, 2-phenylethen-1-yl, naphthylmethyl,2-naphthylethan-1-yl, 2-naphthylethen-1-yl, naphthobenzyl,2-naphthophenylethan-1-yl and the like. Where specific alkyl moietiesare intended, the nomenclature arylalkanyl, arylalkenyl and/orarylalkynyl is used. In preferred embodiments, the arylalkyl group is(C₆-C₁₆) arylalkyl, e.g., the alkanyl, alkenyl or alkynyl moiety of thearylalkyl group is (C₁-C₆) and the aryl moiety is (C₅-C₁₀). Inparticularly preferred embodiments the arylalkyl group is (C₆-C₁₃),e.g., the alkanyl, alkenyl or alkynyl moiety of the arylalkyl group is(C₁-C₃) and the aryl moiety is (C₅-C₁₀).

[0095] “Parent Heteroaromatic Ring System” refers to a parent aromaticring system in which one or more carbon atoms are each independentlyreplaced with the same or different heteroatoms or heteroatomic groups.Typical heteroatoms or heteroatomic groups to replace the carbon atomsinclude, but are not limited to, N, NH, P, O, S, Si, etc. Specificallyincluded within the definition of “parent heteroaromatic ring systems”are fused ring systems in which one or more of the rings are aromaticand one or more of the rings are saturated or unsaturated, such as, forexample, arsindole, benzodioxan, benzofuran, chromane, chromene, indole,indoline, xanthene, etc. Also included in the definition of “parentheteroaromatic ring system” are those recognized rings that includesubstituents, such as benzopyrone. Typical parent heteroaromatic ringsystems include, but are not limited to, arsindole, benzodioxan,benzofuran, benzopyrone, carbazole, β-carboline, chromane, chromene,cinnoline, furan, imidazole, indazole, indole, indoline, indolizine,isobenzofuran, isochromene, isoindole, isoindoline, isoquinoline,isothiazole, isoxazole, naphthyridine, oxadiazole, oxazole, perimidine,phenanthridine, phenanthroline, phenazine, phthalazine, pteridine,purine, pyran, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine,pyrrole, pyrrolizine, quinazoline, quinoline, quinolizine, quinoxaline,tetrazole, thiadiazole, thiazole, thiophene, triazole, xanthene, and thelike.

[0096] “Heteroaryl” by itself or as part of another substituent refersto a monovalent heteroaromatic group having the stated number of ringatoms (i.e., “5-14 membered” means from 5 to 14 ring atoms) derived bythe removal of one hydrogen atom from a single atom of a parentheteroaromatic ring system. Typical heteroaryl groups include, but arenot limited to, groups derived from acridine, arsindole, carbazole,β-carboline, chromane, chromene, cinnoline, furan, imidazole, indazole,indole, indoline, indolizine, isobenzofuran, isochromene, isoindole,isoindoline, isoquinoline, isothiazole, isoxazole, naphthyridine,oxadiazole, oxazole, perimidine, phenanthridine, phenanthroline,phenazine, phthalazine, pteridine, purine, pyran, pyrazine, pyrazole,pyridazine, pyridine, pyrimidine, pyrrole, pyrrolizine, quinazoline,quinoline, quinolizine, quinoxaline, tetrazole, thiadiazole, thiazole,thiophene, triazole, xanthene, and the like. In preferred embodiments,the heteroaryl group is a 5-14 membered heteroaryl, with 5-10 memberedheteroaryl being particularly preferred.

[0097] “Heteroarylalkyl” by itself or as part of another substituentrefers to an acyclic alkyl group in which one of the hydrogen atomsbonded to a carbon atom, typically a terminal or sp³ carbon atom, isreplaced with a heteroaryl group. Where specific alkyl moieties areintended, the nomenclature heteroarylalkanyl, heteroarylalkenyl and/orheterorylalkynyl is used. In preferred embodiments, the heteroarylalkylgroup is a 6-20 membered heteroarylalkyl, e.g., the alkanyl, alkenyl oralkynyl moiety of the heteroarylalkyl is 1-6 membered and the heteroarylmoiety is a 5-14-membered heteroaryl. In particularly preferredembodiments, the heteroarylalkyl is a 6-13 membered heteroarylalkyl,e.g., the alkanyl, alkenyl or alkynyl moiety is 1-3 membered and theheteroaryl moiety is a 5-10 membered heteroaryl.

[0098] “Substituted Alkyl, Aryl, Arylalkyl, Heteroaryl orHeteroarylalkyl” refers to an alkyl, aryl, arylalkyl, heteroaryl orheteroarylalkyl group in which one or more hydrogen atoms is replacedwith another substituent group. Exemplary substituent groups include,but are not limited to, —OR′, —SR′, —NR′R′, —NO₂, —NO, —CN, —CF₃,halogen (e.g., —F, —Cl, —Br and —I), —C(O)R′, —C(O)OR′, —C(O)NR′,—S(O)₂R′, —S(O)₂NR′R′, where each R′ is independently selected from thegroup consisting of hydrogen and (C₁-C₆) alkyl.

[0099] The terms “percentage of sequence identity” and “percentagehomology” are used interchangeably herein to refer to comparisons amongpolynucleotides and polypeptides, and are determined by comparing twooptimally aligned sequences over a comparison window, wherein theportion of the polynucleotide or polypeptide sequence in the comparisonwindow may comprise additions or deletions (i.e., gaps) as compared tothe reference sequence (which does not comprise additions or deletions)for optimal alignment of the two sequences. The percentage may becalculated by determining the number of positions at which the identicalnucleic acid base or amino acid residue occurs in both sequences toyield the number of matched positions, dividing the number of matchedpositions by the total number of positions in the window of comparisonand multiplying the result by 100 to yield the percentage of sequenceidentity. Alternatively, the percentage may be calculated by determiningthe number of positions at which either the identical nucleic acid baseor amino acid residue occurs in both sequences or a nucleic acid base oramino acid residue is aligned with a gap to yield the number of matchedpositions, dividing the number of matched positions by the total numberof positions in the window of comparison and multiplying the result by100 to yield the percentage of sequence identity. Those of skill in theart appreciate that there are many established algorithms available toalign two sequences. Optimal alignment of sequences for comparison canbe conducted, e.g., by the local homology algorithm of Smith & Waterman,Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm ofNeedleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search forsimilarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA85:2444 (1988), by computerized implementations of these algorithms(GAP, BESTFIT, FASTA, and TFASTA in the GCG Wisconsin Software Package),or by visual inspection (see generally, Current Protocols in MolecularBiology, F. M. Ausubel et al., eds., Current Protocols, a joint venturebetween Greene Publishing Associates, Inc. and John Wiley & Sons, Inc.,(1995 Supplement) (Ausubel)). Examples of algorithms that are suitablefor determining percent sequence identity and sequence similarity arethe BLAST and BLAST 2.0 algorithms, which are described in Altschul etal. (1990) J. Mol. Biol. 215: 403-410 and Altschul et al. (1977) NucleicAcids Res. 3389-3402, respectively. Software for performing BLASTanalyses is publicly available through the National Center forBiotechnology Information website. This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence, which either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as, theneighborhood word score threshold (Altschul et al, supra). These initialneighborhood word hits act as seeds for initiating searches to findlonger HSPs containing them. The word hits are then extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Cumulative scores are calculated using, fornucleotide sequences, the parameters M (reward score for a pair ofmatching residues; always >0) and N (penalty score for mismatchingresidues; always <0). For amino acid sequences, a scoring matrix is usedto calculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, M=5, N=-4, and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlength(W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

[0100] While all of the above mentioned algorithms and programs aresuitable for a determination of sequence alignment and % sequenceidentity, for determination of % sequence identity in connection withthe present invention, the BESTFIT or GAP programs in the GCG WisconsinSoftware package (Accelrys, Madison Wis.), using default parametersprovided, are preferred.

[0101] The CL02A3 Compounds

[0102] The CL02A3 compounds of the invention are generally peptidesand/or peptides analogs which, as will be discussed in more detailbelow, are capable of modulating a variety of processes involved in IL-4receptor-mediated isotype switching of B-cells to produce IgE. TheCL02A3 compounds of the invention are generally peptides or peptideanalogs, or pharmaceutically-acceptable salts thereof, that are between8 and 30 amino acid residues in length and include a “core” peptide orpeptide analog, comprising at least 8 consecutive amino acid residues,preferably at least 10 consecutive amino acid residues, more preferablyat least 12 amino acid residues, according to structural formula (II):

X¹—X²˜X³˜X⁴˜X⁵˜X⁶˜X⁷˜X⁸˜X⁹˜X¹⁰˜X¹¹˜X¹²˜X¹³˜X¹⁴˜X¹⁵˜X¹⁶˜X¹⁷˜X¹⁸˜X¹⁹˜X²⁰  (II)

[0103] wherein:

[0104] X¹ is a small aliphatic residue;

[0105] X is a non-polar residue;

[0106] X³ is an aromatic residue, a basic residue or a Ala residue;

[0107] X⁴ is a Gly or Ala residue;

[0108] X⁵ is an aromatic residue, a basic residue or a Ala residue;

[0109] X⁶ is an aromatic residue, a basic residue or a Ala residue;

[0110] X⁷ is an aliphatic residue;

[0111] X⁸ is an aliphatic residue or an aromatic residue;

[0112] X⁹ is a conformationally-constrained residue or a Ala residue;

[0113] X¹⁰ is an aromatic residue or a Ala residue;

[0114] X¹¹ is a Gly or Ala residue;

[0115] X¹² is a non-polar residue;

[0116] X¹³ is an acidic residue or a Ala residue;

[0117] X¹⁴ is a polar residue or a Ala residue;

[0118] X¹⁵ is a Gly or Ala residue;

[0119] X¹⁶ is a cysteine-like residue or a Ala residue;

[0120] X¹⁷ is a hydroxyl-containing residue or a Ala residue;

[0121] X¹⁸ is a hydrophobic residue;

[0122] X¹⁹ is an aliphatic residue; and

[0123] X²⁰ is an aliphatic residue.

[0124] The CL02A3 compounds of the invention include linear, branchedand cyclic peptides and peptide analogs.

[0125] The CL02A3 compounds of the invention and/or the “core” peptidesor peptide analogs of structure (II) are defined, in part, in terms ofamino acids or residues bearing side chains belonging to certaindesignated classes. The definitions of the various classes of aminoacids or residues that define structure (II), and hence the CL02A3compounds of the invention, are as follows:

[0126] “Hydrophilic Amino Acid or Residue” refers to an amino acid orresidue having a side chain exhibiting a hydrophobicity of less thanzero according to the normalized consensus hydrophobicity scale ofEisenberg et al., 1984, J. Mol. Biol. 179:125-142. Genetically encodedhydrophilic amino acids include L-Thr (T), L-Ser (S), L-His (H), L-Glu(E), L-Asn (N), L-Gln (O), L-Asp (D), L-Lys (K) and L-Arg (R).

[0127] “Acidic Amino Acid or Residue” refers to a hydrophilic amino acidor residue having a side chain exhibiting a pK value of less than about6 when the amino acid is included in a peptide or polypeptide. Acidicamino acids typically have negatively charged side chains atphysiological pH due to loss of a hydrogen ion. Genetically encodedacidic amino acids include L-Glu (E) and L-Asp (D).

[0128] “Basic Amino Acid or Residue” refers to a hydrophilic amino acidor residue having a side chain exhibiting a pK value of greater thanabout 6 when the amino acid is included in a peptide or polypeptide.Basic amino acids typically have positively charged side chains atphysiological pH due to association with hydronium ion. Geneticallyencoded basic amino acids include L-His (H), L-Arg (R) and L-Lys (K).

[0129] “Polar Amino Acid or Residue” refers to a hydrophilic amino acidor residue having a side chain that is uncharged at physiological pH,but which has at least one bond in which the pair of electrons shared incommon by two atoms is held more closely by one of the atoms.Genetically encoded polar amino acids include L-Asn (N), L-Gln (Q),L-Ser (S) and L-Thr (T)

[0130] “Hydrophobic Amino Acid or Residue” refers to an amino acid orresidue having a side chain exhibiting a hydrophobicity of greater thanzero according to the normalized consensus hydrophobicity scale ofEisenberg et al., 1984, J. Mol. Biol. 179:125-142. Genetically encodedhydrophobic amino acids include L-Pro (P), L-Ile (I), L-Phe (F), L-Val(V), L-Leu (L), L-Trp (W), L-Met (M), L-Ala (A) and L-Tyr (Y).

[0131] “Aromatic Amino Acid or Residue” refers to a hydrophilic orhydrophobic amino acid or residue having a side chain that includes atleast one aromatic or heteroaromatic ring. The aromatic orheteroaromatic ring may contain one or more substituents such as —OH,—OR″, —SH, —SR″, —CN, halogen (e.g., —F, —Cl, —Br, —I), —NO₂, —NO, —NH₂,—NHR″, —NR″R″, —C(O)R″, —C(O)O⁻, —C(O)OH, —C(O)OR″, —C(O)NH₂, —C(O)NHR″,—C(O)NR″R″ and the like, where each R″ is independently (C₁-C₆) alkyl,substituted (C₁-C₆) alkyl, (C₂-C₆) alkenyl, substituted (C₂-C₆) alkenyl,(C₂-C₆) alkynyl, substituted (C₂-C₆) alkynyl, (C₅-C₁₀) aryl, substituted(C₅-C₁₀) aryl, (C₆-C₁₆) arylalkyl, substituted (C₆-C₁₆) arylalkyl, 5-10membered heteroaryl, substituted 5-10 membered heteroaryl, 6-16 memberedheteroarylalkyl or substituted 6-16 membered heteroarylalkyl.Genetically encoded aromatic amino acids include L-Phe (F), L-Tyr (Y)and L-Trp (W). Although owing to the pKa of its heteroaromatic nitrogenatom L-His (H) is classified above as a basic residue, as its side chainincludes a heteroaromatic ring, it may also be classified as an aromaticresidue.

[0132] “Non-polar Amino Acid or Residue” refers to a hydrophobic aminoacid or residue having a side chain that is uncharged at physiologicalpH and which has bonds in which the pair of electrons shared in commonby two atoms is generally held equally by each of the two atoms (i.e.,the side chain is not polar). Genetically encoded non-polar amino acidsinclude L-Leu (L), L-Val (V), L-Ile (I), L-Met (M) and L-Ala (A).

[0133] “Aliphatic Amino Acid or Residue” refers to a hydrophobic aminoacid or residue having an aliphatic hydrocarbon side chain. Geneticallyencoded aliphatic amino acids include L-Ala (A), L-Val (V), L-Leu (L)and L-Ile (I).

[0134] The amino acid L-Cys (C) is unusual in that it can form disulfidebridges with other L-Cys (C) amino acids or other sulfhydryl- orsulfanyl-containing amino acids. The “cysteine-like residues” includecysteine and other amino acids that contain sulfhydryl moieties that areavailable for formation of disulfide bridges. The ability of L-Cys (C)(and other amino acids with —SH containing side chains) to exist in apeptide in either the reduced free —SH or oxidized disulfide-bridgedform affects whether L-Cys (C) contributes net hydrophobic orhydrophilic character to a peptide. While L-Cys (C) exhibits ahydrophobicity of 0.29 according to the normalized consensus scale ofEisenberg (Eisenberg et al., 1984, supra), it is to be understood thatfor purposes of the present invention L-Cys (C) is categorized as apolar hydrophilic amino acid, notwithstanding the generalclassifications defined above.

[0135] The amino acid Gly (G) is also unusual in that it bears no sidechain on its α-carbon and, as a consequence, contributes only a peptidebond to a particular peptide sequence. Moreover, owing to the lack of aside chain, it is the only genetically-encoded amino acid having anachiral α-carbon. Although Gly (G) exhibits a hydrophobicity of 0.48according to the normalized consensus scale of Eisenberg (Eisenberg etal., 1984, supra), for purposes of the present invention, Gly iscategorized as an aliphatic amino acid or residue.

[0136] Owing in part to its conformationally constrained nature, theamino acid L-Pro (P) is also unusual. Although it is categorized hereinas a hydrophobic amino acid or residue, it will typically occur inpositions near the N- and/or C-termini so as not to deleteriously affectthe structure of the CL02A3 compounds. However, as will be appreciatedby skilled artisans, CL02A3 compounds may include L-Pro (P) or othersimilar “conformationally constrained” residues at internal positions.100851 “Small Amino Acid or Residue” refers to an amino acid or residuehaving a side chain that is composed of a total three or fewer carbonand/or heteroatoms (excluding the α-carbon and hydrogens). The smallamino acids or residues may be further categorized as aliphatic,non-polar, polar or acidic small amino acids or residues, in accordancewith the above definitions. Genetically-encoded small amino acidsinclude Gly, L-Ala (A), L-Val (V), L-Cys (C), L-Asn (N), L-Ser (S),L-Thr (T) and L-Asp (D).

[0137] “Hydroxyl-containing residue” refers to an amino acid containinga hydroxyl (—OH) moiety. Genetically-encoded hydroxyl-containing aminoacids include L-Ser (S) L-Thr (T) and L-Tyr (Y).

[0138] As will be appreciated by those of skill in the art, theabove-defined categories are not mutually exclusive. Indeed, thedelineated category of small amino acids includes amino acids from allof the other delineated categories except the aromatic category. Thus,amino acids having side chains exhibiting two or more physico-chemicalproperties can be included in multiple categories. As a specificexample, amino acid side chains having heteroaromatic moieties thatinclude ionizable heteroatoms, such as His, may exhibit both aromaticproperties and basic properties, and can therefore be included in boththe aromatic and basic categories. The appropriate classification of anyamino acid or residue will be apparent to those of skill in the art,especially in light of the detailed disclosure provided herein.

[0139] While the above-defined categories have been exemplified in termsof the genetically encoded amino acids, the CL02A3 compounds of theinvention are not restricted to the genetically encoded amino acids.Indeed, in addition to the genetically encoded amino acids, the CL02A3compounds of the invention may be comprised, either in whole or in part,of naturally-occurring and/or synthetic non-encoded amino acids. Certaincommonly encountered non-encoded amino acids of which the cycliccompounds of the invention may be comprised include, but are not limitedto: the D-enantiomers of the genetically-encoded amino acids;2,3-diaminopropionic acid (Dpr); α-aminoisobutyric acid (Aib);ε-aminohexanoic acid (Aha); δ-aminovaleric acid (Ava); N-methylglycineor sarcosine (MeGly or Sar); ornithine (Orn); citrulline (Cit);t-butylalanine (Bua); t-butylglycine (Bug); N-methylisoleucine (MeIle);phenylglycine (Phg); cyclohexylalanine (Cha); norleucine (Nle);naphthylalanine (NaI); 2-chlorophenylalanine (Ocf);3-chlorophenylalanine (Mcf); 4-chlorophenylalanine (Pcf);2-fluorophenylalanine (Off); 3-fluorophenylalanine (Mff);4-fluorophenylalanine (Pff); 2-bromophenylalanine (Obf);3-bromophenylalanine (Mbf); 4-bromophenylalanine (Pbf);2--methylphenylalanine (Omf); 3-methylphenylalanine (Mmf);4-methylphenylalanine (Pmf); 2-nitrophenylalanine (Onf);3-nitrophenylalanine (Mnf); 4-nitrophenylalanine (Pnf);2-cyanophenylalanine (Ocf); 3-cyanophenylalanine (Mcf);4-cyanophenylalanine (Pcf); 2-trifluoromethylphenylalanine (Otf);3-trifluoromethylphenylalanine (Mtf); 4-trifluoromethylphenylalanine(Ptf); 4-aminophenylalanine (Paf); 4-iodophenylalanine (Pif);4-aminomethylphenylalanine (Pamf); 2,4-dichlorophenylalanine (Opef);3,4-dichlorophenylalanine (Mpcf); 2,4-difluorophenylalanine (Opff);3,4-difluorophenylalanine (Mpff); pyrid-2-ylalanine (2pAla);pyrid-3-ylalanine (3pAla); pyrid-4-ylalanine (4pAla); naphth-1-ylalanine(1nAla); naphth-2-ylalanine (2nAla); thiazolylalanine (taAla);benzothienylalanine (bAla); thienylalanine (tAla); furylalanine (fAla);homophenylalanine (hPhe); homotyrosine (hTyr); homotryptophan (hTrp);pentafluorophenylalanine (5ff); styrylkalanine (sAla); authrylalanine(aAla); 3,3-diphenylalanine (Dfa); 3-amino-5-phenypentanoic acid (Afp);penicillamine (Pen); 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid(Tic); P-2-thienylalanine (Thi); methionine sulfoxide (Mso);N(w)-nitroarginine (nArg); homolysine (hLys);phosphonomethylphenylalanine (pmPhe); phosphoserine (pSer);phosphothreonine (pThr); homoaspartic acid (hAsp); homoglutanic acid(hGlu); 1-aminocyclopent-(2 or 3)-ene-4 carboxylic acid; pipecolic acid(PA), azetidine-3-carboxylic acid (ACA);1-aminocyclopentane-3-carboxylic acid; allylglycine (aOly);propargylglycine (pgGly); homoalanine (hAla); norvaline (nVal);homoleucine (hLeu), homovaline (hVal); homoisolencine (hIle);homoarginine (hArg); N-acetyl lysine (AcLys); 2,4-diaminobutyric acid(Dbu); 2,3-diaminobutyric acid (Dab); N-methylvaline (MeVal);homocysteine (hCys); homoserine (hSer); hydroxyproline (Hyp) andhomoproline (hPro). Additional non-encoded amino acids of which thecompounds of the invention may be comprised will be apparent to those ofskill in the art (see, e.g., the various amino acids provided in Fasman,1989, CRC Practical Handbook of Biochemistry and Molecular Biology, CRCPress, Boca Raton, Fla., at pp. 3-70 and the references cited therein,all of which are incorporated by reference). These amino acids may be ineither the L- or D-configuration.

[0140] Those of skill in the art will recognize that amino acids orresidues bearing side chain protecting groups may also comprise theCL02A3 compounds of the invention. Non-limiting examples of suchprotected amino acids, which in this case belong to the aromaticcategory, include (protecting groups listed in parentheses), but are notlimited to: Arg(tos), Cys(methylbenzyl), Cys (nitropyridinesulfenyl),Glu(6-benzylester), Gln(xanthyl), Asn(N-δ-xanthyl), His(bom),His(benzyl), His(tos), Lys(fmoc), Lys(tos), Ser(O-benzyl), Thr(O-benzyl) and Tyr(O-benzyl).

[0141] Non-encoding amino acids that are conformationally constrained ofwhich the CL02A3 compounds of the invention may be composed include, butare not limited to, N-methyl amino acids (L-configuration);1-aminocyclopent-(2 or 3)-ene-4-carboxylic acid; pipecolic acid;azetidine-3-carboxylic acid; homoproline (hPro); and1-aminocyclopentane-3-carboxylic acid.

[0142] The classifications of the genetically encoded and certain commonnon-encoded amino acids according to the categories defined above aresummarized in TABLE 1, below. It is to be understood that TABLE 1 is forillustrative purposes only and does not purport to be an exhaustive listof amino acids that can comprise the CL02A3 compounds of the invention.Other amino acids not specifically mentioned herein can be readilycategorized based on their observed physical and chemical properties inlight of the definitions provided herein. TABLE 1 Encoded and CertainCommon Non-Encoded Amino Acid Classifications Classifi- Encoded AminoNon-encoded Amino cation Acids Acids Hydrophobic Aromatic F, Y, W, H f,y, w, h, Phg, Nal, Thi, Tic, Pcf, Off, Mff, Pff, hPhe Non-Polar L, V, I,M, G, A, P l, v, i, m, g, a, p, Bua, Bug, MeIle, Nie, MeVal, Cha, MeGly,Aib Aliphatic A, V, L, I a,v, l, i, Dpr, Aib, Aha, MeGly, Bua, Bug,Mele, Cha, Nle, MeVal Hydrophilic Acidic D, E d, e Basic H, K, R h, k,r, Dpr, Orn, hArg, Paf, Dbu, Dab Polar C, Q, N, S, T c, q, n, s, t, Cit,AcLys, Mso, hSer Small G, A, V, C, N, S, T, D g, a, v, c, n, s, t, d

[0143] In the “core” peptides and peptide analogs of structure (II), thesymbol “˜” between each specified residue X^(n) designates a backboneconstitutive linking moiety. When the CL02A3 compounds of the inventionare peptides, each “˜” between the various X^(n) represents an amide orpeptide linkage of the following polarity: —C(O)—NH—. It is to beunderstood, however, that the CL02A3 compounds of the invention includeanalogs of peptides in which one or more amide or peptide linkages arereplaced with a linkage other than an amide or peptide linkage, such asa substituted amide linkage, an isostere of an amide linkage, or apeptido or amide mimetic linkage. Thus, when used in connection withdefining the various X^(n) comprising the CL02A3 compounds of theinvention, the term “residue” refers to the C_(α) carbon and side chainmoiety(ies) of the designated amino acid or class of amino acid. As aspecific example, defining X¹ as being a “Gly residue” means that X¹ isC_(α)H₂. Defining X¹ as being an “Ala residue” means that X¹ isC_(α)HCH₃ in which the C_(α) carbon is in either the D- orL-configuration. Defining X¹ as being an “A residue” means that X¹ isC_(α)HCH₃ in which the C_(α) carbon is in the L-configuration.

[0144] Substituted amide linkages that may be included in the CL02A3compounds of the invention include, but are not limited to, groups ofthe formula —C(O)NR², where R² is (C₁-C₆) alkyl, (C₅-C₁₀) aryl,substituted (C₅-C₁₀) aryl, (C₆-C₁₆) arylalkyl, substituted (C₆-C₁₆)arylalkyl, 5-10 membered heteroaryl, substituted 5-10 memberedheteroaryl, 6-16 membered heteroarylalkyl or substituted 6-16 memberedheteroarylalkyl. In a specific embodiment, R² is (C₁-C₆) alkanyl,(C₂-C₆) alkenyl, (C₂-C₆) alkynyl or phenyl.

[0145] Isosteres of amides that may be included in the CL02A3 compoundsof the invention generally include, but are not limited to, —NR³—SO—,—NR³—S(O)₂—, —CH₂—CH₂—, —CH═CH—(cis and trans), —CH₂—NH—, —CH₂—S—,—CH₂—O—, —C(O)—CH₂—, —CH(OH)—CH₂—and —CH₂—S(O)₂—, where R³ is hydrogenor R² and R² is as previously defined. These interlinkages may beincluded in the CL02A3 compounds of the invention in either the depictedpolarity or in the reverse polarity. Peptide analogs including suchnon-amide linkages, as well as methods of synthesizing such analogs, arewell-known. See, for example, Spatola, 1983, “Peptide BackboneModifications,” In: Chemistry and Biochemistry of Amino Acids, Peptidesand Proteins, Weinstein, Ed., Marcel Dekker, New York, pp. 267-357(general review); Morley, 1980, Trends Pharm. Sci. 1:463-468; Hudson etal., 1979, Int. J. Prot. Res. 14:177-185 (—CH₂—NH—, —CH₂—CH₂); Spatolaet al., 1986, Life Sci. 38:1243-1249; Spatola, 1983, “Peptide BackboneModifications: the ψ [CH₂S] Moiety as an Amide Bond Replacement,” In:Peptides: Structure and Function V, J. Hruby and D. H. Rich, Eds.,Pierce Chemical Co., Rockford, Ill., pp. 341-344 (—CH₂—S—); Hann, 1982,J. Chem. Soc. Parkin Trans. 1.1:307-314 (—CH═CH—, cis and trans);Almquist et al., 1980, J. Med. Chem. 23:1392-1398 (—C(O)—CH₂—); EuropeanPatent Application EP 45665; Chemical Abstracts CA 97:39405(—CH(OH)—CH₂—); Holladay et al., 1983, Tetrahedron Lett. 24:4401-4404(—CH(OH)—CH₂—); and Hruby, 1982, Life Sci. 31:189-199 (—CH₂—S—).

[0146] Alternatively, one or more amide linkages may be replaced withpeptidomimetic and/or amide mimetic moieties. Non-limiting examples ofsuch moities are described in Olson et al., 1993, J. Med. Chem.36:3039-3049; Ripka & Rich, 1998, Curr. Opin. Chem. Biol. 2:441-452;Borchardt et al., 1997, Adv. Drug. Deliv. Rev. 27:235-256 and thevarious references cited therein.

[0147] While structure (II) contains 20 specified residue positions, itis to be understood that the CL02A3 compounds of the invention maycontain fewer than 20 residues. Indeed, truncated forms of structure(II) containing as few as 8 residues that retain one or more of theutilities described herein are considered to be within the scope of thepresent invention. Truncated forms of the compounds of structure (II)are obtained by deleting one or more residues from either or bothtermini. Preferred truncated forms of structure (II) will contain atleast 10 residues; more preferred truncated forms of structure (II) willcontain at least 12 to 16 residues or more.

[0148] The core peptides or peptide analogs of structure (II) may alsobe extended at one or both termini. Typically, such extensions willrange from about 1 to about 5 residues, but may be even longer, so longas the compound retains one or more of the utilities described herein.For example, one or both termini may be extended by 6, 7, 8, 9, 10 oreven more residues.

[0149] In one embodiment of the invention, the extension has a sequencethat corresponds to a sequence of a signal peptide capable of effectingtransport across membranes, such that the CL02A3 compound is a “fusionpolypeptide.” Such fusion polypeptides are particularly advantageous foradministering to cells CL02A3 compounds of the invention that may notreadily traverse cell membranes. The signal sequence may be fused toeither the N-terminal or C-terminal portion of the CL02A3 compound,depending upon the characteristics of the particular signal sequenceselected. Signal sequences capable of transporting molecules into cellsare well-known in the art. Any of these sequences may be used inconnection with the CL02A3 compounds of the invention. Specific examplesof such sequences include HIV Tat sequences (see, e.g., Fawell et al.,1994, Proc. Natl. Acad. Sci. USA 91:664; Frankel et al., 1988, Cell55:1189; Savion et al., 1981, J. Biol. Chem. 256:1149; Derossi et al.,1994, J. Biol. Chem. 269:10444; Baldin et al., 1990, EMBO J. 9:1511;U.S. Pat. No. 5,804,604; U.S. Pat. No. 5,670,617; and U.S. Pat. No.5,652,122, the disclosures of which are incorporated herein byreference), antennapedia sequences (see, e.g., Garcia-Echeverria et al.,2001, Bioorg. Med. Chem. Lett. 11:1363-1366; Prochiantz, 1999, Ann. NYAcad. Sci. 886:172-179; Prochiantz, 1996, Curr. Opin. Neurobiol.6:629-634; U.S. Pat. No. 6,080,724, and the references cited in all ofthe above, the disclosures of which are incorporated herein byreference) and poly(Arg) or poly(Lys) chains of 5-10 residues.Additional non-limiting examples of specific sequences can be found inU.S. Pat. No. 6,248,558; U.S. Pat. No. 6,043,339; U.S. Pat. No.5,807,746 U.S. Pat. No. 6,251,398; U.S. Pat. No. 6,184,038 and U.S. Pat.No. 6,017,735, the disclosures of which are incorporated herein byreference.

[0150] The terminus of the CL02A3 compounds of the inventioncorresponding to the amino terminus, if present, may be in the “free”form (e.g., H₂N—), or alternatively may be acylated with a group of theformula R²C(O)— or R²S(O)₂—, wherein R² is as previously defined. In oneembodiment, R² is selected from the group consisting of (C₁-C₆) alkyl,(C₅-C₁₀) aryl, (C₆-C₁₆) arylalkyl, 5-10 membered heteroaryl or 6-16membered heteroarylalkyl. In a specific embodiment, the R² group is agroup that facilitates entry of the CL02A3 compound into a cell. Suchgroups are well-known in the art.

[0151] In another embodiment, the amino terminus may be “blocked” with ablocking group designed to impart the CL02A3 compound with specifiedproperties, such as a low antigenicity. Non-limiting examples of suchblocking groups include polyalkylene oxide polymers such as polyethyleneglycol (PEG). A variety of polymers useful for imparting compounds, andin particular peptides and proteins, with specified properties are knownin the art, as are chemistries suitable for attaching such polymers tothe compounds. Specific non-limiting examples may be found in U.S. Pat.Nos. 5,643,575; 5,730,990; 5,902,588; 5,919,455; 6,113,906; 6,153,655;and 6,177,087, the disclosures of which are incorporated herein byreference.

[0152] Of course, skilled artisans will appreciate that any of thesetransport-effecting, acylating and/or blocking groups may also beattached to a side chain moiety of a CL02A3 compound. Residues havingappropriate functionalities for attaching such groups will be apparentto those of skill in the art, and include, by way of example and notlimitation, Cys, Lys, Asp and Glu.

[0153] The terminus of the CL02A3 compounds corresponding to theC-terminus, if present, may be in the form of an underivatized carboxylgroup, either as the free acid or as a salt, such as a sodium,potassium, calcium, magnesium salt or other salt of an inorganic ororganic ion, or may be in the form of a derivatized carboxyl, such as anester, thioester or amide. Such derivatized forms of the compounds maybe prepared by reacting a CL02A3 compound having a carboxyl terminuswith an appropriate alcohol, thiol or amine. Suitable alcohols, thiolsor amines include, by way of example and not limitation, alcohols of theformula R²OH, thiols of the formula R²SH and amines of the formulaR²NH₂, R²R²NH or NH₃, where each R² is, independently of the others, aspreviously defined.

[0154] The C-terminus may also include transport-effecting or otherblocking groups, such as those described above.

[0155] As will be recognized by skilled artisans, the various X^(n)residues comprising the CL02A3 compounds of the invention may be ineither the L- or D-configuration about their C_(α) carbons. In oneembodiment, all of the Ca carbons of a particular CL02A3 compound are inthe same configuration. In some embodiments of the invention, the CL02A3compounds comprise specific chiralities about one or more C_(α)carbon(s) and/or include non-peptide linkages at specified locations soas to impart the CL02A3 compound with specified properties. For example,it is well-known that peptides composed in whole or in part of D-aminoacids are more resistant to proteases than their corresponding L-peptidecounterparts. Thus, in one embodiment, the CL02A3 compounds are peptidescomposed in whole or in part of D-amino acids. Alternatively, CL02A3compounds having good stability against proteases may include peptideanalogs including peptide linkages of reversed polarity at specifiedpositions. For example, CL02A3 compounds having stability againsttryptic-like proteases include peptide analogs having peptide linkagesof reversed polarity before each L-Arg or L-Lys residue; CL02A3compounds having stability against chymotrypsin-like proteases includepeptide analogs having peptide linkages of reversed polarity before eachsmall and medium-sized L-aliphatic residue or L-non-polar residue. Inanother embodiment, CL02A3 compounds having stability against proteasesinclude peptide analogs composed wholly of peptide bonds of reversedpolarity. Other embodiments having stability against proteases will beapparent to those of skill in the art. Additional specific embodimentsof the CL02A3 compounds of the invention are described below.

[0156] The CL02A3 compounds of the invention can be in a linear form ora cyclic form, with or without branching. The cyclic forms can becyclized via the terminal groups or via side chain groups on internal orterminal residues, through covalent or non-covalent linkages. Additionallinking groups may also be present to facilitate cyclization.

[0157] In one specific embodiment, the CL02A3 compounds of the inventionare 20-residue peptides or peptide analogs according to structuralformula (III):

Z³-X¹˜X²˜X³˜X⁴˜X⁵˜X⁶˜X⁷˜X⁸˜X⁹˜X¹⁰˜X¹¹˜X¹²˜X¹³˜X¹⁴˜X¹⁵˜X¹⁶˜X¹⁷˜X¹⁸˜X⁹˜X²⁰-Z⁴  (III)

[0158] wherein:

[0159] each X¹ through X²⁰ is as previously defined for structure (II);

[0160] Z³ is H₂N—, R⁴HN— or R⁴C(O)NH—;

[0161] Z⁴ is —C(O)O⁻, —C(O)OR⁴, —C(O)NHR⁴ or —C(O)NH₂;

[0162] each R⁴ is independently (C₁-C₆) alkyl or (C₁-C₆) alkanyl;

[0163] each “˜” is independently an amide linkage, a substituted amidelinkage or an isostere of an amide linkage; and

[0164] each “-” represents a bond.

[0165] In another specific embodiment, the CL02A3 compounds of theinvention are compounds according to structural formula (III) in whicheach “˜” is an amide linkage.

[0166] In yet another specific embodiment, the CL02A3 compounds of theinvention are compounds according to structural formula (III) in which:

[0167] X¹ is L-Ala;

[0168] X² is L-Met;

[0169] X³ is L-His;

[0170] X⁴ is Gly;

[0171] X⁵ is L-His;

[0172] X⁶ is L-His;

[0173] X⁷ is a small aliphatic residue;

[0174] X⁸ is an aromatic or a small aliphatic residue;

[0175] X⁹ is L-Pro;

[0176] X¹⁰ is L-Trp;

[0177] X¹¹ is Gly;

[0178] X¹² is L-Met;

[0179] X¹³ is L-Glu;

[0180] X¹⁴ is L-Gln;

[0181] X¹⁵ is Gly;

[0182] X¹⁶ is L-Cys;

[0183] X¹⁷ is L-Thr;

[0184] X¹⁸ is L-Pro;

[0185] X¹⁹ is a small aliphatic residue; and

[0186] X²⁰ is a small aliphatic residue.

[0187] Preferably, X⁷ is Gly or L-Ala, X⁸ is L-Trp or L-Ala, X¹⁹ isL-Leu or L-Ala and/or X²⁰ is L-Ala or Gly.

[0188] In another specific embodiment, the CL02A3 compounds of theinvention include compounds according to structural formula IV, andvariants of such compounds of formula IV in which 1, 2, 3, or 4,preferably 1 or 2, of the amino acid residues set forth in IV arereplaced by another amino acid selected from the same class (asdescribed herein) as the original amino acid or by an Ala residue:

Z¹-A˜M˜H˜G˜H˜H˜G˜W˜P˜W˜G˜M˜EQ˜G˜C˜T˜P˜L˜G-Z²  (IV)

[0189] wherein “-”, “˜”, Z¹ and Z² are as defined previously for formula(1).

[0190] In still another specific embodiment, the CL02A3 compounds of theinvention are selected from the group consisting of CL02A3 wt(AMHGHHGWPWGMEQGCTPLG SEQ ID NO:1), CL02A3LG (AMHGHHGWPWGMEQGCTPAA SEQID NO:2), CL02A3GW (AMHGHHAAPWGMEQGCTPLG SEQ ID NO:3), CL02A3TP(AMHGHHGWPWGMEQGCAALG SEQ ID NO:4), CL02A3EQ (AMHGHHGWPWGMAAGCTPLG SEQID NO:5), CL02A3HG (AMAAHHGWPWGMEQGCTPLG SEQ ID NO:6), CL02A3GC(AMHGHHGWPWGMEQAATPLG SEQ ID NO:7), CL02A3HH (AMHGAAGWPWGMEQGCTPLG SEQID NO:8), CL02A3PW (AMHGHHGWAAGMEQGCTPLG SEQ ID NO:9), and analogs andprotease-resistant analogs thereof. Preferred CL02A3 compounds areselected from CL02A3 wt, CL02A3LG, CL02A3GW, CL02A3TP, and CL02A3EQ.

[0191] Active CL02A3 compounds of the invention are those that modulate,and in particular inhibit or downregulate, IL-4 induced IgE productionand/or accumulation and/or processes associated therewith. The CL02A3compounds of the invention may be assessed for such activity in anystandard assay that assesses the ability of a compound to modulate IL-4induced IgE production and/or accumulation. For example, a CL02A3compound of the invention may be administered to a human or animalB-cell (e.g., primary B cells from blood, tonsils, spleens and otherlymphoid tissues) stimulated with IL-4 (available from Pharmingen,Hamburg, Germany) and anti-CD40 mabs (available from Ancell Corporation,Bayport Minn.) and the amount of IgE produced measured, for example, byan ELISA technique, such as the ELISA technique described in Worm etal., 1998, Blood 92:1713. The ELISA technique can use, for example,murine anti human IgE, biotinylated anti human IgE and streptavidinbiotinylated horseradish peroxidase complex. Specific ELISA assays andtechniques that may be used are provided in the Examples section.Particular active CL02A3 compounds include without limitation CL02A3 wt,CL02A3LG, CL02A3TP, CL02A3EQ, CL02A3HG, CL02A3GC, CL02A3HH, CL02A3PW,and CL02A3GW.

[0192] For CL02A3 compounds that readily traverse cell membranes, thecompound may be administered to the cell by contacting the cell with thecompound. CL02A3 compounds composed wholly of genetically-encoded aminoacids that do not readily traverse cell membranes may be administered tothe cell using well-known delivery techniques. In one embodiment, suchCL02A3 compounds may be administered using well-known retroviral vectorsand infection techniques pioneered by Richard Mulligan and DavidBaltimore with Psi-2 lines and analogous retroviral packaging systemsbased upon NIH 3T3 cells (see Mann et al., 1993, Cell 33:153-159, thedisclosure of which is incorporated herein by reference). Suchhelper-defective packaging cell lines are capable of producing all ofthe necessary trans proteins (gag, pol and env) required for packaging,processing, reverse transcribing and integrating genomes. Those RNAmolecules that have in cis the ψ packaging signal are packaged intomaturing retrovirions. Virtually any of the art-known retroviral vectorsand/or transfection systems may be used. Specific non-limiting examplesof suitable transfection systems include those described in WO 97/27213;WO 97/27212; Choate et al., 1996, Human Gene Therapy 7:2247-2253;Kinsella et al., 1996, Human Gene Therapy 7:1405-1413; Hofmann et al.,1996, Proc. Natl. Acac. Sci. USA 93:5185-5190; Kitamura et al., 1995,Proc. Natl. Acac. Sci. USA 92:9146-9150; WO 94/19478; Pear et al., 1993,Proc. Natl. Acac. Sci. USA 90:8392-8396; Mann et al., 1993, Cell33:153-159 and the references cited in all of the above, the disclosuresof which are incorporated herein by reference. Specific non-limitingexamples of suitable retroviral vector systems include vectors basedupon murine stem cell virus (MSCV) as described in Hawley et al., 1994,Gene Therapy 1:136-138; vectors based upon a modified MFG virus asdescribed in Rivere et al., 1995, Genetics 92:6733; pBABE as describedin WO 97/27213 and WO 97/27212; and the vectors depicted in FIG. 11 ofWO 01/34806, the disclosures of which are incorporated herein byreference. Other suitable vectors and/or transfection techniques arediscussed in connection with gene therapy administration, infra.

[0193] A specific assay for assessing IL-4 induced IgE production thatmay be used to assay CL02A3 compounds of the invention is described inWorm et al., 2001, Int. Arch. Allergy Immunol. 124:233-236. Generally, aCL02A3 compound modulates IL-4 induced IgE production if it yields anincrease or decrease in measured IgE levels of at least about 25% ascompared to control cells (i.e., cells activated with IL-4+anti-CD40Mabs but not exposed to the CL02A3 compound). CL02A3 compounds thatincrease IL-4 induced IgE production are IgE agonists whereas CL02A3compounds that decrease IL-4 induced IgE production are IgE antagonists.Skilled artisans will appreciate that CL02A3 compounds that inhibitgreater levels of IL-4 induced IgE production, for example on the orderof 50%, 60%, 70%, 80%, 90%, or even more as compared to control cells,are particularly desirable. Thus, while compounds that inhibit at leastabout 25% of IL-4 induced IgE production as compared to control cellsare active, compounds that inhibit at least about 50%, 75% or even moreIL-4 induced IgE production as compared to control cells are preferred.

[0194] In another embodiment, CL02A3 compounds may be assayed for theability to modulate IL-4 induced transcription of a germline ε promoter.Generally, such assays involve administering a CL02A3 compound to anIL-4 induced cell comprising an IL-4 inducible germline ε promoter andassessing the amount of gene expression (i.e. transcription) downstreamof the ε promoter. Depending upon the ability of the CL02A3 compound totraverse cell membranes, it may be administered to the cell bycontacting the cell with the compound or (for peptide compounds) via theretroviral transfection techniques described supra. The amount of thedownstream gene expression may be assessed at the mRNA level, forexample by quantifying the amount of a downstream transcription productproduced, or at the translation level, for example by quantifying theamount of a downstream translation product produced. In one embodiment,the germline ε promoter is operably linked to a reporter gene thatencodes a protein that produces an observable and/or detectable signal,such as a fluorescent protein. Specific examples of suitable assays forassessing CL02A3 compounds for the ability to modulate germline εtranscription are described in U.S. Pat. No. 5,958,707, WO 01/34806, WO99/58663, commonly owned copending application Ser. No. 09/712,821,filed Nov. 13, 2000 and commonly owned copending application Ser. No.09/076,624, filed May 12, 1998, the disclosures of which areincorporated herein by reference. Generally, a CL02A3 compound modulatesgermline ε transcription if it yields an increase or decrease inmeasured downstream expression of at least about 25% as compared tocontrol cells activated with IL-4 but not exposed to the CL02A3compound. CL02A3 compounds that increase downstream expression are IL-4agonists, whereas compounds that decrease (i.e. inhibit) downstreamexpression are IL-4 antagonists. Skilled artisans will appreciate thatCL02A3 compounds that inhibit greater levels of IL-4 induced germline εtranscription, for example on the order of 50%, 60%, 70%, 80%, 90%, oreven more as compared to control cells, are particularly desirable.Thus, while compounds that inhibit at least about 25% of IL-4 inducedgermline ε transcription as compared to control cells are active,compounds that inhibit at least about 50%, 75% or even more IL-4 inducedgermline ε transcription as compared to control cells are preferred. Inone embodiment of the invention, active CL02A3 compounds are those thatexhibit a reporter ratio of ≧1.1 in the A5T4 reporter line screeningassay described in the examples section. In general, the “reporterratio” is the ratio of the signal from a reporter under control of the εpromoter in the absence of a CL02A3 compound to that in the presence ofthe CL02A3 compound. In particular, for screening in the A5T4 reporterline that has been transformed with the inhibitor peptide vector, thereporter ratio can be determined from the ratio of the GFP fluorescenceof IL-4 stimulated cells in the presence of doxycycline or tetracycline(i.e., when expression of the peptide is repressed) to the GFPfluorescence of IL-4 stimulated cells in the absence of doxycycline ortetracycline.

[0195] As mentioned previously, B-cells initially produce IgD and IgMimmunoglobulins and, when induced by the proper cytokines, produce IgEs.B-cells can be induced to produce other types of immunoglobulins, suchas IgGs and IgAs, as well. For example, in the presence of the cytokineinterleukin-2 (IL-2), B-cells produce IgG1; in the presence of acombination of IL-2 and TGF-β, B-cells produce IgA. In many situations,it is desirable to selectively modulate (increase or decrease) theproduction of a single immunoglobulin isotype, as such specificitypermits the ability to treat or prevent diseases associated with theproduction and/or accumulation of the specified immunoglobulin isotypewithout affecting the immune system generally. Thus, in one embodiment,the CL02A3 compounds specifically modulate IL-4 induced germline Etranscription or IL-4 induced IgE production and/or accumulation. By“specific” is meant that the CL02A3 compound modulates IL-4 induced IgEproduction and/or accumulation or IL-4 induced germline E transcriptionbut does not significantly affect the production and/or accumulation ofanother immunoglobulin, or transcription of the promoter of another Igisotype. In a particular embodiment, a CL02A3 compound specificallyinhibits IL-4 induced germline ε transcription or IL-4 induced IgEproduction or accumulation. Such CL02A3 compound does not significantlyinhibit production and/or accumulation of another Ig isotype, ortranscription of another Ig isotype promoter, if the observed inhibitionof the other Ig isotype in an appropriate assay is on the order of 10%or less as compared to control cells. Such specificity may be withrespect to a single Ig isotype, or may be with respect to one or more Igisotypes. For example, a CL02A3 compound may be assessed for specificityby assaying its ability to inhibit, for example, IgA production and/oraccumulation or to inhibit germline ε transcription in assays similar tothose described above, except that the cells are activated witheffectors suitable for IgA switching and synthesis and amount of IgAproduced or the amount of expression downstream of a germline ε promoteris assessed. Specific, non-limiting examples of CL02A3 compounds thatspecifically inhibit IL-4 induced IgE production and/or IL-4 inducedgermline ε transcription include peptides CL02A3 wt, CL02A3LG, CL02A3TP,CL02A3EQ, CL02A3HG, CL02A3GC, CL02A3HH, CL02A3PW, and CL02A3GW.

[0196] As will be discussed in more detail below, it has been discoveredthat the ability of certain CL02A3 compounds to inhibit IL-4 induced IgEproduction and/or 1L-4 induced germline ε transcription is mediated bybinding a CLLD8 protein. Accordingly, CL02A3 compounds may also beassessed for activity based upon their ability to bind a CLLD8 proteinusing, for example, any of the protein binding assays described infra.Generally, active CL02A3 compounds are those having a binding constant(Kd) on the order of 10 mM or less, with Kds in the range of 100 μM, 10μM, 1 μM, 100 nM, 10 nM, 1 nM or even lower, being preferred.Alternatively, active CL02A3 compounds are those that compete forbinding a CLLD8 protein with another active CL02A3 compound. In aspecific embodiment, the CL02A3 compound competes for binding a CLLD8protein with peptides CL02A3 wt, CL02A3LG, CL02A3LG, CL02A3TP, CL02A3EQ,CL02A3HG, CL02A3GC, CL02A3HH, CL02A3PW, or CL02A3GW. The ability of aCL02A3 compound to compete for binding a CLLD8 protein with anotherCL02A3 compound may be assessed using conventional competitive bindingassay techniques. Generally, active CL02A3 compounds are those thatexhibit an IC₅₀ in the range of 1 mM or lower, with IC₅₀s in the rangeof 100 μM, 10 μM, 1 μM, 100 nM, 10 nM, 1 nM or even lower, in suchcompetitive binding assays being preferred.

[0197] Chemical Synthesis of the CL02A3 Compounds

[0198] CL02A3 compounds of the invention may be prepared using standardtechniques of organic synthesis. CL02A3 compounds that are peptides maybe prepared using conventional step-wise solution or solid phasesynthesis (see, e.g., Chemical Approaches to the Synthesis of Peptidesand Proteins, Williams et al., Eds., 1997, CRC Press, Boca Raton Fla.,and references cited therein; FMOC Solid Phase Peptide Synthesis: APractical Approach, Chan & White, Eds., 2000, IRL Press, Oxford,England, and references cited therein).

[0199] Alternatively, CL02A3 compounds, may be prepared by way ofsegment condensation, as described, for example, in Liu et al., 1996,Tetrahedron Lett. 37(7):933-936; Baca et al., 1995, J. Am. Chem. Soc.117:1881-1887; Tam et al., 1995, Int. J. Peptide Protein Res.45:209-216; Schnolzer and Kent, 1992, Science 256:221-225; Liu and Tam,1994, J. Am. Chem. Soc. 116(10):4149-4153; Liu and Tam, 1994, Proc.Natl. Acad. Sci. USA 91:6584-6588; Yamashiro and Li, 1988, Int. J.Peptide Protein Res. 31:322-334. The condensation technique isparticularly useful for synthesizing CL02A3 compounds comprising Glyresidues. Other methods useful for synthesizing the CL02A3 compounds ofthe invention are described in Nakagawa et al., 1985, J. Am. Chem. Soc.107:7087-7092. CL02A3 compounds that are peptide analogs may besynthesized using the various methods described in the references citedin connection with amide isosteres and amide and peptidomimetics, supra.

[0200] CL02A3 compounds containing N- and/or C-terminal blocking groupscan be prepared using standard techniques of organic chemistry. Forexample, methods for acylating the N-terminus of a peptide or amidatingor esterifying the C-terminus of a peptide are well-known in the art.Modes of carrying other modifications at the N- and/or C-terminus willbe apparent to those of skill in the art, as will modes of protectingany side-chain functionalities as may be necessary to attach terminalblocking groups.

[0201] Formation of disulfide linkages, if desired, is generallyconducted in the presence of mild oxidizing agents. Chemical oxidizingagents may be used, or the compounds may simply be exposed toatmospheric oxygen to effect these linkages. Various methods are knownin the art, including those described, for example, by Tam et al., 1979,Synthesis 955-957; Stewart et al., 1984, Solid Phase Peptide Synthesis,2d Ed., Pierce Chemical Company Rockford, Ill.; Ahmed et al., 1975, J.Biol. Chem. 250:8477-8482; and Pennington et al., 1991 Peptides 1990164-166, Giralt and Andreu, Eds., ESCOM Leiden, The Netherlands. Anadditional alternative is described by Kamber et al., 1980, Helv. Chim.Acta 63:899-915. A method conducted on solid supports is described byAlbericio, 1985, Int. J. Peptide Protein Res. 26:92-97. Any of thesemethods may be used to form disulfide linkages in the CL02A3 compoundsof the invention.

[0202] Cyclic peptides may be prepared or may result from the formationof single or multiple disulfide bonds, other side-chains or head-to-tailcyclizations, either directly or by way of an optional linker. Thecyclic peptides may be prepared using any art-known techniques for thepreparation of cyclic peptides and cyclic peptide analogs. For example,the peptide or peptide analog may be prepared in linear or non-cyclizedform using conventional solution or solid phase peptide and/or peptideanalog syntheses and cyclized using standard chemistries. The linearpolypeptides can be cyclized with a linking group between the twotermini, between one terminus and the side chain of an amino acid in thepeptide or peptide derivative, or between the side chains to two aminoacids in the peptide or peptide derivative. Suitable procedures forsynthesizing the peptide and peptide analogs described herein, as wellas suitable chemistries for cyclizing such compounds, are well known inthe art. For references related to synthesis of cyclic peptides thereader is referred to Tam et al., 2000, Biopolymers 52:311-332; Camameroet al, 1998, Angew. Chem. Intl. Ed. 37: 347-349; Tam et al., 1998, Prot.Sci. 7:1583-1592; Jackson et al., 1995, J. Am. Chem. Soc. 117:819-820;Dong et al., 1995, J. Am. Chem. Soc. 117:2726-2731; Ishida et al., 1995,J. Org. Chem. 60:5374-5375; WO 95/33765, published Jun. 6, 1995; Xue andDeGrado, 1994, J. Org. Chem. 60(4):946-952; Jacquier et al., 1991, In:Peptides 1990 221-222, Giralt and Andreu, Eds., ESCOM Leiden, TheNetherlands; Schmidt and Neubert, 1991, In: Peptides 1990 214-215,Giralt and Andreu, Eds., ESCOM Leiden, The Netherlands; Toniolo, 1990,Int. J. Peptide Protein Res. 35:287-300; Ulysse et al., 1995, J. Am.Chem. Soc. 117:8466-8467; Durr et al., 1991, Peptides 1990 216-218,Giralt and Andreu, Eds., ESCOM Leiden, The Netherlands; Lender et al.,1993, Int. J. Peptide Protein Res. 42:509-517; Boger and Yohannes, 1990,J. Org. Chem. 55:6000-6017; Brady et al., 1979, J. Org. Chem.4(18):3101-3105; Spatola et al., 1986, J. Am. Chem. Soc. 108:825-831;Seidel et al., 1991, In: Peptides 1990 236-237, Giralt and Andreu, Eds.,ESCOM Leiden, The Netherlands; Tanizawa et al., 1986, Chem. Phar, Bull.34(10):4001-4011; Goldenburg & Creighton, 1983, J. Mol. Biol.165:407-413; WO 92/00995 and WO 94/15958. These methods may be routinelyadapted to synthesize the cyclic compounds of the invention and areincorporated into this application by reference.

[0203] Recombinant Synthesis of the CL02A3 Compounds

[0204] If the CL02A3 compound is composed entirely ofgenetically-encoded amino acids, or a portion of it is so composed, thepeptide or the relevant portion may also be synthesized usingconventional recombinant genetic engineering techniques.

[0205] For recombinant production, a polynucleotide sequence encodingthe peptide is inserted into an appropriate expression vehicle, i.e., avector which contains the necessary elements for the transcription andtranslation of the inserted coding sequence, or in the case of an RNAviral vector, the necessary elements for replication and translation.The expression vehicle is then transfected into a suitable target cellwhich will express the peptide. Depending on the expression system used,the expressed peptide is then isolated by procedures well-established inthe art. Methods for recombinant protein and peptide production are wellknown in the art (see, e.g., Sambrook et al, 1989, Molecular Cloning ALaboratory Manual, Cold Spring Harbor Laboratory, N.Y.; and Ausubel etal., 1989, Current Protocols in Molecular Biology, Greene PublishingAssociates and Wiley Interscience, N.Y. each of which is incorporated byreference herein in its entirety.)

[0206] To increase efficiency of production, the polynucleotide can bedesigned to encode multiple units of the peptide separated by enzymaticcleavage sites—either homopolymers (repeating peptide units) orheteropolymers (different peptides strung together) can be engineered inthis way. The resulting polypeptide can be cleaved (e.g., by treatmentwith the appropriate enzyme) in order to recover the peptide units. Thiscan increase the yield of peptides driven by a single promoter. In apreferred embodiment, a polycistronic polynucleotide can be designed sothat a single mRNA is transcribed which encodes multiple peptides (i.e.,homopolymers or heteropolymers) each coding region operatively linked toa cap-independent translation control sequence; e.g., an internalribosome entry site (IRES). When used in appropriate viral expressionsystems, the translation of each peptide encoded by the mRNA is directedinternally in the transcript; e.g., by the IRES. Thus, the polycistronicconstruct directs the transcription of a single, large polycistronicmRNA which, in turn, directs the translation of multiple, individualpeptides. This approach eliminates the production and enzymaticprocessing of polyproteins and may significantly increase yield ofpeptide driven by a single promoter.

[0207] Polynucleotides capable of generating or expressing certaincyclic peptide embodiments of the compounds of the invention may beprepared in vitro and/or in vivo. Polypeptides may be prepared frompolynucleotides to generate or express the cyclic peptides utilizing thetrans splicing ability of split inteins. Methods for making suchpolynucleotides to yield cyclic peptides are known in the art and aredescribed, for example, in WO 01/66565, WO 00/36093; U.S. PatentApplication No. 60/358,827, entitled “Cyclic Peptides and Analogs Usefulto Treat Allergies”, filed on Feb. 21, 2002, the disclosures of whichare incorporated herein by reference.

[0208] A variety of host-expression vector systems may be utilized toexpress the peptides described herein. These include, but are notlimited to, microorganisms such as bacteria transformed with recombinantbacteriophage DNA or plasmid DNA expression vectors containing anappropriate coding sequence; yeast or filamentous fungi transformed withrecombinant yeast or fungi expression vectors containing an appropriatecoding sequence; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing an appropriate codingsequence; plant cell systems infected with recombinant virus expressionvectors (e.g., cauliflower mosaic virus or tobacco mosaic virus) ortransformed with recombinant plasmid expression vectors (e.g., Tiplasmid) containing an appropriate coding sequence; or animal cellsystems.

[0209] The expression elements of the expression systems vary in theirstrength and specificities. Depending on the host/vector systemutilized, any of a number of suitable transcription and translationelements, including constitutive and inducible promoters, may be used inthe expression vector. For example, when cloning in bacterial systems,inducible promoters such as pL of bacteriophage lambda, plac, ptrp, ptac(ptrp-lac hybrid promoter) and the like may be used; when cloning ininsect cell systems, promoters such as the baculovirus polyhedronpromoter may be used; when cloning in plant cell systems, promotersderived from the genome of plant cells (e.g., heat shock promoters; thepromoter for the small subunit of RUBISCO; the promoter for thechlorophyll a/b binding protein) or from plant viruses (e.g., the 35SRNA promoter of CaMV; the coat protein promoter of TMV) may be used;when cloning in mammalian cell systems, promoters derived from thegenome of mammalian cells (e.g., metallothionein promoter) or frommammalian viruses (e.g., the adenovirus late promoter; the vacciniavirus 7.5 K promoter) may be used; when generating cell lines thatcontain multiple copies of expression product, SV40-, BPV- and EBV-basedvectors may be used with an appropriate selectable marker.

[0210] In cases where plant expression vectors are used, the expressionof sequences encoding the peptides of the invention may be driven by anyof a number of promoters. For example, viral promoters such as the 35SRNA and 19S RNA promoters of CaMV (Brisson et al., 1984, Nature310:511-514), or the coat protein promoter of TMV (Takamatsu et al.,1987, EMBO J. 6:307-311) may be used; alternatively, plant promoterssuch as the small subunit of RUBISCO (Coruzzi et al., 1984, EMBO J.3:1671-1680; Broglie et al., 1984, Science 224:838-843) or heat shockpromoters, e.g., soybean hspl7.5-E or hspl 7.3-B (Gurley et al., 1986,Mol. Cell. Biol. 6:559-565) may be used. These constructs can beintroduced into plant cells using Ti plasmids, Ri plasmids, plant virusvectors, direct DNA transformation, microinjection, electroporation,etc. For reviews of such techniques see, e.g., Weissbach & Weissbach,1988, Methods for Plant Molecular Biology, Academic Press, N.Y., SectionVIII, pp. 421-463; and Grierson & Corey, 1988, Plant Molecular Biology,2d Ed., Blackie, London, Ch. 7-9.

[0211] In one insect expression system that may be used to produce thepeptides of the invention, Autographa californica, nuclear polyhidrosisvirus (AcNPV) is used as a vector to express the foreign genes. Thevirus grows in Spodoptera frugiperda cells. A coding sequence may becloned into non-essential regions (for example the polyhedron gene) ofthe virus and placed under control of an AcNPV promoter (for example,the polyhedron promoter). Successful insertion of a coding sequence willresult in inactivation of the polyhedron gene and production ofnon-occluded recombinant virus (i.e., virus lacking the proteinaceouscoat coded for by the polyhedron gene). These recombinant viruses arethen used to infect Spodoptera frugiperda cells in which the insertedgene is expressed (e.g., see Smith et al., 1983, J. Virol. 46:584; U.S.Pat. No. 4,215,051). Further examples of this expression system maybefound in Current Protocols in Molecular Biology, Vol. 2, Ausubel et al.,eds., Greene Publish. Assoc. & Wiley Interscience.

[0212] In mammalian host cells, a number of viral based expressionsystems may be utilized. In cases where an adenovirus is used as anexpression vector, a coding sequence may be ligated to an adenovirustranscription/translation control complex, e.g., the late promoter andtripartite leader sequence. This chimeric gene may then be inserted inthe adenovirus genome by in vitro or in vivo recombination. Insertion ina non-essential region of the viral genome (e.g., region E1 or E3) willresult in a recombinant virus that is viable and capable of expressingpeptide in infected hosts. (e.g., see Logan & Shenk, 1984, Proc. Natl.Acad. Sci. USA 81:3655-3659). Alternatively, the vaccinia 7.5 K promotermay be used, (see, e.g., Mackett et al., 1982, Proc. Natl. Acad. Sci.USA 79:7415-7419; Mackett et al., 1984, J. Virol. 49:857-864; Panicaliet al., 1982, Proc. Natl. Acad. Sci. USA 79:4927-4931).

[0213] Other expression systems for producing CL02A3 peptides of theinvention will be apparent to those having skill in the art.

[0214] Purification of CL02A3 Compounds

[0215] The CL02A3 compounds of the invention can be purified byart-known techniques such as reverse phase chromatography highperformance liquid chromatography, ion exchange chromatography, gelelectrophoresis, affinity chromatography and the like. The actualconditions used to purify a particular compound will depend, in part, onsynthesis strategy and on factors such as net charge, hydrophobicity,hydrophilicity, etc., and will be apparent to those having skill in theart.

[0216] For affinity chromatography purification, any antibody whichspecifically binds the compound may be used. For the production ofantibodies, various host animals, including but not limited to rabbits,mice, rats, etc., may be immunized by injection with a compound. Thecompound may be attached to a suitable carrier, such as BSA, by means ofa side chain functional group or linkers attached to a side chainfunctional group. Various adjuvants may be used to increase theimmunological response, depending on the host species, including but notlimited to Freund's (complete and incomplete), mineral gels such asaluminum hydroxide, surface active substances such as lysolecithin,pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanin, dinitrophenol, and potentially useful human adjuvants suchas BCG (bacilli Calmette-Guerin) and Corynebacterium parvum.

[0217] Monoclonal antibodies to a compound may be prepared using anytechnique which provides for the production of antibody molecules bycontinuous cell lines in culture. These include, but are not limited to,the hybridoma technique originally described by Kohler & Milstein, 1975,Nature 256:495-497 and/or Kaprowski, U.S. Pat. No. 4,376,110; the humanB-cell hybridoma technique described by Kosbor et al., 1983, ImmunologyToday 4:72 and/or Cote et al., 1983, Proc. Natl. Acad. Sci. USA80:2026-2030; and the EBV-hybridoma technique described by Cole et al.,1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp.77-96. In addition, techniques developed for the production of “chimericantibodies” (Morrison et al., 1984, Proc. Natl. Acad. Sci. USA81:6851-6855; Neuberger et al., 1984, Nature 312:604-608; Takeda et al,1985, Nature 314:452-454; Boss, U.S. Pat. No. 4,816,397; Cabilly, U.S.Pat. No. 4,816,567) by splicing the genes from a mouse antibody moleculeof appropriate antigen specificity together with genes from a humanantibody molecule of appropriate biological activity can be used. Or“humanized” antibodies can be prepared (see, e.g., Queen, U.S. Pat. No.5,585,089). Alternatively, techniques described for the production ofsingle chain antibodies (see, e.g., U.S. Pat. No. 4,946,778) can beadapted to produce compound-specific single chain antibodies.

[0218] Antibody fragments which contain deletions of specific bindingsites may be generated by known techniques. For example, such fragmentsinclude but are not limited to F(ab′)2 fragments, which can be producedby pepsin digestion of the antibody molecule and Fab fragments, whichcan be generated by reducing the disulfide bridges of the F(ab′)2fragments. Alternatively, Fab expression libraries may be constructed(Huse et al., 1989, Science 246:1275-1281) to allow rapid and easyidentification of monoclonal Fab fragments with the desired specificityfor the peptide of interest.

[0219] The antibody or antibody fragment specific for the desiredpeptide can be attached, for example, to agarose, and theantibody-agarose complex is used in immunochromatography to purifypeptides of the invention. See, Scopes, 1984, Protein Purification:Principles and Practice, Springer-Verlag New York, Inc., N.Y.,Livingstone, 1974, Methods In Enzymology: Immunoaffinity Chromatographyof Proteins 34:723-731.

[0220] As will be recognized by skilled artisans, the above methods mayalso be used to prepare anti-CLLD8 protein antibodies. Such anti-CLLD8protein antibodies may be used in the various methods described herein,for example, to inhibit IL-4 induced isotype switching and/or IgEproduction and/or to inhibit IL-4 induced germline ε transcription. Suchantibodies may also be used in the various therapeutic methods describedherein.

[0221] Screening Assays

[0222] The present inventors have discovered that the IgE regulatoryeffects of three CL02A3 compounds of the invention, peptides CL02A3 wt,CL02A3GW and CL02A3LG, are mediated by a CLLD8 protein. Specifically,the present inventors have discovered that these three CL02A3 compoundsbind a human CLLD8 protein in yeast two hybrid (YTH) assays in a mannerthat correlates with their ability to inhibit IL-4 induced germline εtranscription in in vitro cellular assays.

[0223] CLLD8 is a recently described human protein, of unknown function,that has been suggested to be involved in methylation-mediatedtranscriptional repression (Mabuchi et al. Cancer Res. (2001) 61:2870).The CLLD8 coding region was identified during an analysis of the 13q14chromosomal region, the region of the most common structural aberrationin B-cell chronic lymphocytic leukemia (B-CLL) (Mabuchi et al. 2001,supra). CLLD8 protein contains a methyl-CpG binding domain (MBD) and abifurcated SET domain with adjacent cysteine-rich region (preSETdomain). The SET domain, which was first identified in three Drosophilachromosomal regulators, is now known to be present in manytranscriptional regulators from different species (Hobert et al. Mech.Dev. (1996) 55:171). The SET protein family is divided into foursubgroups based on sequence identity within the SET domains: E[z], TRX,ASHI and Su(var)3-9. CLLD8 belongs to the Su(var)3-9 subgroup, showinghighest sequence homology to the SETDB1 protein (Harte et al. Cytogenet.Cell Genet (1999) 84:83). In addition to CLLD8, this subgroup includeshuman G9A (Milner et al., (1993) Biochem. J. 290:811), and human SUV39H1(Aagaard et al. (1999) EMBO J. 18:1923) and SETDB1 (Harte et al.Cytogenet. Cell Genet. (1999) 84:83). The cellular function of theSETDB1 protein is not yet known. As CLLD8 has both a MBD and a SETdomain, it is thought that it might be a histone methyltransferase thatmay be associated with methylation-mediated transcriptional repressionand has been suggested to have a role in leukemogenesis based on itspostulated activity and its chromosomal location. However, no cellularactivity for CLLD8 has yet been identified. The present inventors arethe first to discover a link between the human CLLD8 protein andmodulation of the IL-4 signaling cascade involved in the production ofIgE, and in particular to IL-4 induced isotype switching of B-cells toproduce IgE.

[0224] This significant discovery enables, for the first time, theability to use a CLLD8 protein as a “surrogate” analyte in simplebinding assays to screen for and/or identify compounds involved in IL-4induced IgE regulation. Such compounds are useful in the treatmentand/or prevention of diseases caused by or associated with IgEproduction and/or accumulation, such as anaphylactic hypersensitivity orallergic reactions and/or symptoms associated with such reactions,allergic rhinitis, allergic conjunctivitis, systemic mastocytosis, hyperIgE syndrome, and IgE gammopathies, atopic disorders such as atopicdermatitis, atopic eczema and atopic asthma, and B-cell lymphoma.

[0225] Thus, the invention also provides methods and kits useful foridentifying compounds having specified utilities. In specificembodiments, the methods and kits may be used to identify compounds thatinhibit IL-4 induced IgE production and/or accumulation, compounds thatinhibit IL-4 induced isotype switching of B-cells to produce IgE,compounds that inhibit IL-4 induced germline ε transcription, and/orcompounds useful to treat or prevent diseases caused by or associatedwith IgE production and/or accumulation, such as those described above.

[0226] “CLLD8 proteins” useful in the screening methods and kits of theinvention include human CLLD8, a particular example of which can befound in the NCBI protein sequence database at accession #NP_(—)114121.1, shown in FIG. 10 as SEQ ID NO: 18 and allelic andspecies variants thereof, as well as fragments and fusions thereof thatbind to the CL02A3 compounds, in particular peptide CL02A3 wt.Typically, such proteins will have polypeptide sequences that share atleast about 80% identity at the amino acid level with one of the knownisotypes of CLLD8s. Preferably, the CLLD8 protein employed in themethods of the present invention will have at least 85%, 90%, 95% oreven higher % identity with one of the known isotypes of CLLD8. Specificexamples of CLLD8s suitable for use in the methods and kits of theinvention include CLLD8s derived from humans (“hCLLD8”), for example,the CLLD8 protein described by Mabuchi et al. (2001, supra) as well asthe various corresponding mammalian homologs thereof (for example,rodent, rat, mouse, rabbit, canine, simian, etc.). The amino acidsequences of these various CLLD8s, as well as the sequences of nucleicacid molecules encoding these CLLD8s, are known in the art and can befound in the following references and/or NCBI (GenBank) entries: humanCLLL8 NP_(—)114121, gi:13994282; NM_(—)031915.

[0227] As will be recognized by skilled artisans, mutants and/orfragments of a CLLD8 protein may also be used in the assays and kits ofthe invention. Mutants and fragments that are useful in this regard areones that retain the ability ti bind an active CL02A3 compound,preferably peptide CL02A3 wt, peptideCL02A3GW or peptide CL02A3LG.Suitable fragments include CLLD8 proteins that are truncated at the N-and/or C-terminus by one or more amino acids, typically by about 1 to10-20 amino acids, although fragments truncated by more amino acids maybe used, provided the fragments bind an active CL02A3 compound.Additionally, mutants or fragments that substantially retain one or moreof the biological activities of the CLLD8 protein are useful in theassays and kits of the present invention. By “substantially retain” ismeant that the mutant or fragment has at least 10% of the biologicalactivity of the CLLD8 protein as measured by any conventional assay ofactivity; preferably, the mutant or fragment has at least 50% of thebiological activity of CLLD8.

[0228] CLLD8 protein mutants useful in the methods and kits of theinvention include conservative mutants in which one or more amino acidsis replaced with another amino acid of the same class, as defined abovein connection with the description of the CL02A3 compounds. Of courseCLLD8 protein mutants including non-conservative substitutions may alsobe used, so long as the particular mutant binds an active CL02A3compound and/or substantially retains CLLD8 activity. Thus, unlessindicated otherwise, the expression “CLLD8 protein” as used hereinspecifically includes such mutants and/or fragments in addition to thefull-length wild-type proteins.

[0229] The CLLD8 proteins may be obtained using conventional recombinantand purification techniques or may be isolated directly from the naturalsource. For example, any of the recombinant techniques discussed suprain connection with the CL02A3 compounds may be used to produce a CLLD8protein suitable for use in methods and kits of the invention. Suchrecombinantly-produced CLLD8 proteins may be isolated using affinitychromatography (for example, with an anti-CLLD8 protein antibody or bysynthesizing a CLLD8 fusion protein or an epitope-tagged protein) orother conventional techniques. Other techniques for obtaining CLLD8proteins for use in the methods and kits of the invention will beapparent to those of skill in the art.

[0230] Any screening technique known in the art for determining whethercompounds bind one another can be used to screen for compounds that binda CLLD8 protein. The compounds screened can range from small organicmolecules to large polymers and biopolymers, and can include, by way ofexample and not limitation, small organic compounds, saccharides,carbohydrates, polysaccharides, lectins, peptides and analogs thereof,polypeptides, proteins, antibodies, oligonucleotides, polynucleotides,nucleic acids, etc. In one embodiment, the candidate compounds screenedare small organic molecules having a molecular weight in the range ofabout 100-2500 daltons. Such candidate molecules will often comprisecyclical structures composed of carbon atoms or mixtures of carbon atomsand one or more heteroatoms and/or aromatic, polyaromatic,heteroaromatic and/or polyaromatic structures. The candidate agents mayinclude a wide variety of functional group substituents. In oneembodiment, the substituent(s) are independently selected from the groupof substituents known to interact with proteins, such as, for example,amine, carbonyl, hydroxyl and carboxyl groups.

[0231] The candidate compounds may be screened on a compound-by-compoundbasis or, alternatively, using one of the myriad library techniquescommonly employed in the art. For example, synthetic combinatorialcompound libraries, natural products libraries and/or peptide librariesmay be screened using the assays of the invention to identify compoundsthat bind a CLLD8 protein. The candidate compounds may be assessed forthe ability to bind a CLLD8 protein per se, or they may be assessed forthe ability to competitively bind a CLLD8 protein in the presence of anactive CL02A3 compound of the invention, such as peptide CL02A3 wt,peptide CL02A3GW, or peptide CL02A3LG, or another compound thatcompetitively binds a CLLD8 protein in the presence of an active CL02A3compound of the invention. These competitive binding assays can identifycompounds that bind the CLLD8 protein at approximately the same site asthe active CL02A3 compound. Myriad techniques for carrying outcompetitive binding assays are known in the art. Any of these techniquesmay be employed in the present invention.

[0232] Such binding experiments may be conducted wholly in solution or,alternatively, either the CLLD8 protein or the candidate compound may beimmobilized on a solid support. For example, the CLLD8 protein or thecandidate compound may be attached to a glass or other bead or a solidsurface such as, for example, the bottom of a petri dish. Theimmobilization may be mediated by non-covalent interactions or bycovalent interactions. Methods for immobilizing myriad types ofcompounds and proteins on solid supports are well-known. Any of thesemethods may be used to immobilize the CLLD8 protein and/or candidatecompound on solid supports.

[0233] The binding assays may employ a purified CLLD8 protein or,alternatively, the assays may be carried out with nucleosol and/orcytosol fractions from cells that express the CLLD8 protein, eitherendogenously or recombinantly.

[0234] Whether carried out in solution or with an immobilized CLLD8protein or candidate compound, the CLLD8 protein and candidate compoundare typically contacted with one another under conditions conducive tobinding. Although the actual conditions used can vary, typically thebinding assays are carried out under physiological conditions. Theconcentrations of CLLD8 protein and candidate compound used will dependupon, among other factors, whether the CLLD8 protein or candidatecompound is immobilized or free in solution, the binding affinities ofcandidate compounds, etc. Actual concentrations suitable for aparticular assay will be apparent to those of skill in the art.

[0235] In many embodiments of the kits and assays of the invention itmay be convenient to employ a labeled CLLD8 protein and/or labeledcandidate compound. For example, in one convenient embodiment, bindingis assessed by contacting an immobilized candidate compound with alabeled CLLD8 protein and assaying for the presence of immobilizedlabel. For such embodiments, the label may be a direct label, i.e., alabel that itself is detectable or produces a detectable signal, or itmay be an indirect label, i.e., a label that is detectable or produces adetectable signal in the presence of another compound. The method ofdetection will depend upon the labeled used, and will be apparent tothose of skill in the art.

[0236] Examples of suitable direct labels include radiolabels,fluorophores, chromophores, chelating agents, particles,chemiluminescent agents and the like. Suitable radiolabels include, byway of example and not limitation, ³H, ¹⁴C, ³²P, ³⁵S, ³⁶Cl, ⁵⁷Co, ⁵⁸Co,⁵⁹Fe, ⁹⁰Y, ¹²⁵I, ¹³¹I and ¹⁸⁶Re. Suitable fluorophores include, by wayof example and not limitation, fluorescein, rhodamine, phycoerythrin,Texas red, free or chelated lanthamide series salts such as Eu³+ and themyriad fluorophores available from Molecular Probes Inc., Eugene, Oreg.Examples of suitable colored labels include, by way of example and notlimitation, metallic sol particles, for example, gold sol particles suchas those described by Leuvering (U.S. Pat. No. 4,313,734); dye soleparticles such as described by Gribnau et al. (U.S. Pat. No. 4,373,932)and May et al. (WO 88/08534); dyed latex such as those described Snyder(EP 0 280 559 and 0 281 327) and dyes encapsulated in liposomes asdescribed by Campbell et al. (U.S. Pat. No. 4,703,017). Other directlabels that may be used will be apparent to those of skill in the art.

[0237] Examples of suitable indirect labels include enzymes capable ofreacting with or interacting with a substrate to produce a detectablesignal (such as those used in ELISA and EMIT immunoassays), ligandscapable of binding a labeled moiety, and the like. Suitable enzymesuseful as indirect labels include, by way of example and not limitation,alkaline phosphatase, horseradish peroxidase, lysozyme,glucose-6-phosphate dehydrogenase, lactate dehydrogenase and urease. Theuse of these enzymes in ELISA and EMIT immunoassays is described indetail in Engvall, 1980, Methods in Enzymology, 70:419-439 and U.S. Pat.No. 4,857,453.

[0238] Methods of labeling proteins and compounds with a variety oflabels such as those described above are well-known. Any of thesemethods may be used to label CLLD8 proteins and/or candidate compounds.For example, a CLLD8 protein may be labeled with a fluorophore such asfluorescein by incubating the CLLD8 protein with, for example,fluorescein isothiocyanate, using conventional techniques.Alternatively, a CLLD8 protein (or a candidate compound produced byrecombinant techniques) can be labeled metabolically by culturing cellsthat express the CLLD8 protein in the presence of culture mediumsupplemented with a metabolic label, such as, by way of example and notlimitation, [³⁵S]-methionine, one or more [¹⁴C]-labeled amino acids, oneor more [¹⁵N]-labeled amino acids and/or [³H]-labeled amino acids (withthe tritium substituted at non-labile positions).

[0239] In one embodiment of the invention, candidate compounds may bescreened for the ability to bind a CLLD8 protein using an affinitychromatography technique. For example, a CLLD8 protein may be attachedto a chromatography resin according to standard techniques to create aCLLD8 protein affinity resin and this CLLD8 protein affinity resin usedto identify compounds that bind the resin. Alternatively, the candidatecompound could be bound to the resin and the resin used to determinewhether it binds a CLLD8 protein. In another alternative embodiment, anactive CL02A3 compound of the invention may by attached to thechromatography resin. This CL02A3 affinity resin may then be used tobind a CLLD8 protein and the bound complex used to identify compoundsthat compete for binding the CLLD8 protein with the active CL02A3compound, typically by washing the resin with a candidate compound anddetermining whether the candidate compound disrupts the CLLD8protein-CL02A3 compound complex by assaying for the release of CLLD8protein from the resin.

[0240] Although candidate compounds may be screened for the ability tobind a CLLD8 protein on a compound-by-compound basis, it may be moreconvenient to screen large numbers of candidate compounds simultaneouslyusing one of the many library screening methodologies known in the art.One art-known approach uses recombinant bacteriophage to produce largelibraries of peptides which can then be screened in a variety of formatsfor binding to a CLLD8 protein. Using such phage methods, very largelibraries of candidate peptides can be constructed (e.g., 10⁶-10⁸peptides) and screened for binding with a CLLD8 protein. Methods forconstructing and screening such “phage display” libraries are described,for example, in Scott & Smith, 1990, Science 249:386-390; Cwirla et al.,1990, Proc. Natl. Acad. Sci. 87:6378-6382; 1990); Devlin et al., 1990,Science 249:404-406 (1990); U.S. Pat. No. 5,427,908; U.S. Pat. No.5,432,018; U.S. Pat. No. 5,580,717 and U.S. Pat. No. 5,723,286, thedisclosures of which are incorporated herein by reference. Othernon-limiting examples of recombinant library methodologies that may beused in connection with the assays of the invention are described inU.S. Pat. No. 6,156,571; U.S. Pat. No. 6,107,059 and U.S. Pat. No.5,733,731, the disclosures of which are incorporated herein byreference.

[0241] A second art-known approach uses chemical methods to synthesizelibraries of compounds, such as small organic compounds, peptides and/orpeptide analogs, attached to beads or wafers that can then beconveniently screened for binding with a CLLD8 protein. The librariesmay be encoded or non-encoded. Methods of synthesizing such immobilizedlibraries, as well as methods of screening the libraries are described,for example, in Houghten, 1985, Proc. Natl. Acad. Sci. USA 82:5131-5735;Geysen et al., 1986, Molecular Immunology 23:709-715; Geysen et al,1987, J. Immunologic Method 102:259-274; Frank & Doring, 1988,Tetrahedron 44:6031-6040; Fodor et al., 1991, Science 251:767-773; Furkaet al., 1988, 4th International Congress of Biochemistry, Volume 5,Abstract FR:013; Furka, 1991, Int. J. Peptide Protein Res. 37:487-493;Frank, 1992, Tetrahedron 48:9217-9232; Needels et al., 1993, Proc. Natl.Acad. Sci. USA 90:10700-10704; DeWitt et al., 1993, Proc. Natl. Acad.Sci. USA 90:6909-6913; Frank et al., 1993, Biorg. Med. Chem. Lett.3:425-430; Ohlmeyer et al., 1993, Proc. Natl. Acad. Sci. USA90:10922-10926; WO 92/00252; WO 9428028; U.S. Pat. No. 6,329,143; U.S.Pat. No. 6291,183; U.S. Pat. No. 5,885,837; U.S. Pat. No. 5,424,186;U.S. Pat. No. 5,384,261; U.S. Pat. No. 6,165,717; U.S. Pat. No.6,143,497; U.S. Pat. No. 6,140,493; U.S. Pat. No. 5,789,162; U.S. Pat.No. 5,770,358; U.S. Pat. No. 5,708,153; U.S. Pat. No. 5,639,603; U.S.Pat. No. 5,541,061; U.S. Pat. No. 5,525,735; U.S. Pat. No. 5,525,734;U.S. Pat. No. 6,261,776; U.S. Pat. No. 6,239,273; U.S. Pat. No.5,846,839; U.S. Pat. No. 5,770,455; U.S. Pat. No. 5,770,157; U.S. Pat.No. 5,609,826; U.S. Pat. No. 6,001,579; U.S. Pat. No. 5,968,736; U.S.Pat. No. 5,962,337; U.S. Pat. No. 5,789,172; U.S. Pat. No. 5,721,099;U.S. Pat. No. 5,565,324; U.S. Pat. No. 5,010,175; and U.S. Pat. No.4,631,211, the disclosures of which are incorporated herein byreference. For reviews of some of these techniques, see Ellman et al.,1996, Account, Chem. Res. 29:132-143; Gallop et al., 1994, J. Med. Chem.37:1233-1251; Gordon et al., 1994, J. Med. Chem. 37:1385-1401.Non-limiting examples of solid-phase chemical synthesis strategies andconditions useful for synthesizing combinatorial libraries of smallorganic and other compounds may be found in Bunin, 1998, TheCombinatorial Index, Academic Press, London, England (see, e.g., Chapter1-5) and Hermkens et al., 1996, Tetrahedron 52:4527-4554, as well as thereferences cited therein, the disclosures of which are incorporatedherein by reference.

[0242] Another art-known approach utilizes solution-phase chemicalsynthesis techniques to synthesize libraries of compounds, such as, forexample, libraries of small organic compounds, which may then bescreened in the assays of the invention. Methods for synthesizing andscreening such solution-phase libraries are well-known and aredescribed, for example, in Bunin, 1998, The Combinatorial Index,Academinc Press, England (see, e.g., Chapter 6); WO 95/02566; U.S. Pat.No. 5,962,736; U.S. Pat. No. 5,766,481; U.S. Pat. No. 5,736,412 and U.S.Pat. No. 5,712,171, and the references cited therein; the disclosures ofwhich are incorporated herein by reference. Additional review articles,references, patents and books describing myriad techniques forsynthesizing and screening libraries of compounds for the ability tobind another compound such as a CLLD8 protein can be found at Lebl &Leblova: Dynamic Database of References in Molecular Diversity, Internethttp://www.5z.com (see especially the diversity information pages athttp://www.5z.com/divinfo).

[0243] Once a candidate compound that binds the CLLD8 protein has beenidentified, further assays may be carried out to characterize thebinding characteristics of the compound, for example, to determine itsbinding affinity, dissociation constant (Kd), on- and/or off-rates,etc., using well-known techniques. For example, binding affinities canbe determined using saturation kinetics and Scatchard analysis. Forsaturation kinetics, the binding assay can be performed with increasingconcentrations of the candidate compound, which is typically labeledwith, for example, a radiolabel. Competitive binding experiments with anactive CL02A3 or other active compound, for example peptide CL02A3 wt,peptide CL02A3GW or peptide CL02A3LG, can be carried out with increasingconcentrations of unlabeled candidate compound and a fixed concentrationof labeled (for example radiolabled) active CL02A3 or other compound.

[0244] An alternative method for characterizing receptor/ligand bindingcharacteristics of a plurality of compounds in parallel that may beadapted for use in connection with the invention is described in U.S.Pat. No. 5,324,633.

[0245] In one embodiment of the invention, the candidate compoundsidentified will have a dissociation constant (Kd) for CLLD8 protein onthe order of 1 mM, 100 μM, 10 μM, 1 μM, 100 nM, 10 nM, 1 nM or evenlower. In another embodiment of the invention, the candidate compoundsidentified will exhibit an IC₅₀ in a competitive binding assay with anactive CL02A3 compound of the invention or another compound thatcompetes for binding a CLLD8 protein with an active CL02A3 compound ofthe invention on the order of 1 mM, 100 μM, 10 μM, 1 μM, 100 nM, 10 nM,1 μM or even lower. In this context, the IC₅₀ represents theconcentration of candidate compound that displaces 50% of the boundCL02A3 or other compound. Suitable assays for measuring such IC₅₀s arewell-known.

[0246] If desired, the ability of identified candidate compounds tomodulate or regulate IL-4 induced IgE production and/or processesassociated therewith can be confirmed in in vitro assays, such as thosepreviously described in connection with the CL02A3 compounds.

[0247] Knowledge of the interaction surface between a CL02A3 compound ofthe invention such as peptide CL02A3 wt, peptide CL02A3GW and/or peptideCL02A3LG and a CLLD8 protein, and in particular the CLLD8 protein aminoacids involved in binding the CL02A3 compound, can also provide usefulinformation for identifying compounds that bind a CLLD8 protein.Identification and screening of CLLD8 protein binding compounds isfurther facilitated by determining structural features of a CLLD8protein-CL02A3 compound complex, e.g., using X-ray crystallography,neutron diffraction, nuclear magnetic resonance spectrometry or anyother techniques for structure determination. These techniques providefor the rational design or in silico identification of compounds thatbind a CLLD8 protein.

[0248] Candidate compounds identified using the screening assays of theinvention may be agonists or antagonists of the CLLD8 protein. Thus, theidentified compounds may bind to the CLLD8 protein without activating itor may inhibit one or more biological activities of CLLD8 protein(antagonist) or, alternatively, the identified compounds may activateone or more biological activities of the CLLD8 protein (agonists). Thesefunctional assays also provide an indirect means of assessing whether acandidate compound binds a CLLD8 protein. Thus, the method of theinvention may employ functional assays of CLLD8 activity to determinewhether a candidate compound binds CLLD8 protein. Observation of agonistor antagonist activity indicates the candidate compound binds the CLLD8protein. Although the in vivo activity of the CLLD8 protein has not yetbeen determined, based on the presence of the SET and preSET domains,CLLD8 most likely has a methyltransferase activity. CLLD8 protein alsocontains a methyl-CpG binding domain that is likely to mediate bindingto methylated DNA. Methyltransferase activity and methylated DNA bindingactivity of the CLLD8 protein can thus be determined and used for afunctional assay for identifying agonists and antagonists.Methyltransferase assays are well known in the art and can be carriedout as described in, for example, Res et al. 2000 Nature 406:593. DNAbinding assays, for example to methylated DNA, can be carried out bymethods that are well known in the art, for example as described in Nanet al. 1993 Nucleic Acids Res. 21:4886, and the references citedtherein.

[0249] Kits

[0250] The invention also provides kits for carrying out the variousscreening assays and methods of the invention. Such kits will typicallyinclude a CLLD8 protein and a compound that competes for binding to theCLLD8 protein, such as an active CL02A3 compound. The CLLD8 proteinand/or compound may be labeled or unlabeled. The kit may further includeadditional components useful for carrying out the assays and methods.Non-limiting examples of such additional components include labels,labeling reagents, binding buffers, etc. The kit may also includeinstructions teaching its methods of use. In one embodiment, the kitcomprises a CLLD8 protein and a compound selected from among peptideCL02A3 wt, peptide CL02A3LG, peptide CL02A3GW, peptide CL02A3TP, peptideCL02A3EQ, peptide CL02A3HG, peptide CL02A3GC, peptide CL02A3HH, peptideCL02A3PW, or an analog thereof.

[0251] Uses of the CL02A3 Compounds and Identified Compounds

[0252] As discussed previously, the active CL02A3 compounds of theinvention and/or the active CLLD8 protein-binding compounds identifiedby the above-described screening methods (referred to collectively as“active compounds”), can be used in a variety of in vitro, in vivo andex vivo applications to regulate or modulate processes involved with theproduction and/or accumulation of IgE. For example, the active compoundscan be used to modulate, and in particular inhibit, any or all of thefollowing processes in vitro, in vivo or ex vivo: IgE production and/oraccumulation; the IL-4 receptor-mediated signaling cascade leading toisotype switching and/or production of IgE; IL-4 induced switching ofB-cells to produce IgE, IL-4 mediated IgE production; and IL-4 inducedgermline E transcription. In a specific embodiment of the invention, theactive compounds may be used to treat or prevent diseases characterizedby, caused by or associated with production and/or accumulation of IgE.Such treatments may be administered to animals in veterinary contexts orto humans. Diseases that are characterized by, caused by or associatedwith IgE production and/or accumulation, and that can therefore betreated or prevented with the active compounds include, by way ofexample and not limitation, anaphylactic hypersensitivity or allergicreactions and/or symptoms associated with such reactions, allergicrhinitis, allergic conjunctivitis, systemic mastocytosis, hyper IgEsyndrome, and IgE gammopathies, atopic disorders such as atopicdermatitis, atopic eczema and atopic asthma, and B-cell lymphoma.

[0253] When used to treat or prevent such diseases, the active compoundsmay be administered singly, as mixtures of one or more active compoundsor in mixture or combination with other agents useful for treating suchdiseases and/or symptoms associated with such diseases. The activecompounds may also be administered in mixture or in combination withagents useful to treat other disorders or maladies, such as steroids,membrane stabilizers, 5LO inhibitors, leukotriene synthesis and receptorinhibitors, IgE receptor inhibitors, β-agonists, tryptase inhibitors andantihistamines, to name a few. The active compounds may be administeredper se or as pharmaceutical compositions.

[0254] Pharmaceutical compositions comprising the active compounds ofthe invention may be manufactured by means of conventional mixing,dissolving, granulating, dragee-making levigating, emulsifying,encapsulating, entrapping or lyophilization processes. The compositionsmay be formulated in conventional manner using one or morephysiologically acceptable carriers, diluents, excipients or auxiliarieswhich facilitate processing of the active compounds into preparationswhich can be used pharmaceutically. The actual pharmaceuticalcomposition administered will depend upon the mode of administration.Virtually any mode of administration may be used, including, for exampletopical, oral, systemic, inhalation, injection, transdermal, etc.

[0255] The active compound may be formulated in the pharmaceuticalcompositions per se, or in the form of a pharmaceutically acceptablesalt. As used herein, the expression “pharmaceutically acceptable salt”means those salts which retain substantially the biologicaleffectiveness and properties of the active compound and which is notbiologically or otherwise undesirable. Such salts may be prepared frominorganic and organic acids and bases, as is well-known in the art.Typically, such salts are more soluble in aqueous solutions than thecorresponding free acids and bases.

[0256] For topical administration, the active compound(s) may beformulated as solutions, gels, ointments, creams, suspensions, etc. asare well-known in the art.

[0257] Systemic formulations include those designed for administrationby injection, e.g., subcutaneous, intravenous, intramuscular,intrathecal or intraperitoneal injection, as well as those designed fortransdermal, transmucosal oral or pulmonary administration.

[0258] Useful injectable preparations include sterile suspensions,solutions or emulsions of the active compound(s) in aqueous or oilyvehicles. The compositions may also contain formulating agents, such assuspending, stabilizing and/or dispersing agent. The formulations forinjection may be presented in unit dosage form, e.g., in ampules or inmultidose containers, and may contain added preservatives.

[0259] Alternatively, the injectable formulation may be provided inpowder form for reconstitution with a suitable vehicle, including butnot limited to sterile pyrogen free water, buffer, dextrose solution,etc., before use. To this end, the active compound(s) may dried by anyart-known technique, such as lyophilization, and reconstituted prior touse.

[0260] For transmucosal administration, penetrants appropriate to thebarrier to be permeated are used in the formulation. Such penetrants areknown in the art.

[0261] For oral administration, the pharmaceutical compositions may takethe form of, for example, tablets or capsules prepared by conventionalmeans with pharmaceutically acceptable excipients such as binding agents(e.g., pregelatinised maize starch, polyvinylpyrrolidone orhydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystallinecellulose or calcium hydrogen phosphate); lubricants (e.g., magnesiumstearate, talc or silica); disintegrants (e.g., potato starch or sodiumstarch glycolate); or wetting agents (e.g., sodium lauryl sulfate). Thetablets may be coated by methods well known in the art with, forexample, sugars or enteric coatings.

[0262] Liquid preparations for oral administration may take the form of,for example, elixirs, solutions, syrups or suspensions, or they may bepresented as a dry product for constitution with water or other suitablevehicle before use. Such liquid preparations may be prepared byconventional means with pharmaceutically acceptable additives such assuspending agents (e.g., sorbitol syrup, cellulose derivatives orhydrogenated edible fats); emulsifying agents (e.g., lecithin oracacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethylalcohol or fractionated vegetable oils); and preservatives (e.g., methylor propyl-p-hydroxybenzoates or sorbic acid). The preparations may alsocontain buffer salts, flavoring, coloring and sweetening agents asappropriate. Preparations for oral administration may be suitablyformulated to give controlled release of the active compound.

[0263] For buccal administration, the compositions may take the form oftablets or lozenges formulated in conventional manner.

[0264] For rectal and vaginal routes of administration, the activecompound(s) may be formulated as solutions (for retention enemas)suppositories or ointments containing conventional suppository basessuch as cocoa butter or other glycerides.

[0265] For administration by inhalation, the active compound(s) can beconveniently delivered in the form of an aerosol spray from pressurizedpacks or a nebulizer, with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof e.g. gelatin for use in an inhaler or insufflator may be formulatedcontaining a powder mix of the compound and a suitable powder base suchas lactose or starch.

[0266] For prolonged delivery, the active compound(s) can be formulatedas a depot preparation, for administration by implantation; e.g.,subcutaneous, intradermal, or intramuscular injection. Thus, forexample, the active ingredient may be formulated with suitable polymericor hydrophobic materials (e.g., as an emulsion in an acceptable oil) orion exchange resins, or as sparingly soluble derivatives; e.g., as asparingly soluble salt.

[0267] Alternatively, transdermal delivery systems manufactured as anadhesive disc or patch which slowly releases the active compound(s) forpercutaneous absorption may be used. To this end, permeation enhancersmay be used to facilitate transdermal penetration of the activecompound(s). Suitable transdermal patches are described in for example,U.S. Pat. No. 5,407,713.; U.S. Pat. No. 5,352,456; U.S. Pat. No.5,332,213; U.S. Pat. No. 5,336,168; U.S. Pat. No. 5,290,561; U.S. Pat.No. 5,254,346; U.S. Pat. No. 5,164,189; U.S. Pat. No. 5,163,899; U.S.Pat. No. 5,088,977; U.S. Pat. No. 5,087,240; U.S. Pat. No. 5,008,110;and U.S. Pat. No. 4,921,475.

[0268] Alternatively, other pharmaceutical delivery systems may beemployed. Liposomes and emulsions are well-known examples of deliveryvehicles that may be used to deliver active compounds(s). Certainorganic solvents such as dimethylsulfoxide (DMSO) may also be employed,although usually at the cost of greater toxicity.

[0269] The pharmaceutical compositions may, if desired, be presented ina pack or dispenser device which may contain one or more unit dosageforms containing the active compound(s). The pack may, for example,comprise metal or plastic foil, such as a blister pack. The pack ordispenser device may be accompanied by instructions for administration.

[0270] Gene Therapy

[0271] As will be recognized by skilled artisans, active compound(s)that are peptides composed wholly of genetically-encoded amino acids maybe administered utilizing well-known gene therapy techniques. Accordingto such techniques, a gene encoding the active compound may beintroduced either in vivo, ex vivo, or in vitro in a viral vector. Suchvectors include an attenuated or defective DNA virus, such as but notlimited to, herpes simplex virus (HSV), papillomavirus, Epstein Barrvirus (EBV), adenovirus, adeno-associated virus (AAV), and the like.Defective viruses, which entirely or almost entirely lack viral genes,are preferred.

[0272] Defective virus is not infective after introduction into a cell.Use of defective viral vectors allows for administration to cells in aspecific, localized area, without concern that the vector can infectother cells. For example, in the treatment of the various diseasesdescribed herein, lymphocyte B-cells can be specifically targeted.Examples of particular vectors include, but are not limited to, adefective herpes virus I (HSV1) vector (Kaplitt et al., 1991, Molec.Cell. Neurosci. 2:320-330), an attenuated adenovirus vector, such as thevector described by Stratford-Perricaudet et al., 1992, J. Clin. Invest.90:626-630 and a defective adeno-associated virus vector (Samulski etal., 1987, J. Virol. 61:3096-3101; Samulski et al., 1989, J. Virol.63:3822-3828).

[0273] Preferably, for in vitro administration, an appropriateimmunosuppressive treatment is employed in conjunction with the viralvector, e.g., adenovirus vector, to avoid immuno-deactivation of theviral vector and transfected cells. For example, immunosuppressivecytokines, such as interleukin-12 (IL-12), interferon-γ (IFN-γ), oranti-CD4 antibody, can be administered to block humoral or cellularimmune responses to the viral vectors (see, e.g., Wilson, 1995, Nat.Med. 1(9):887-889). In addition, it is advantageous to employ a viralvector that is engineered to express a minimal number of antigens.

[0274] In another embodiment the gene can be introduced in a retroviralvector, e.g., as described in Anderson et al., U.S. Pat. No. 5,399,346;Mann et al., 1983, Cell 33:153; Temin et al., U.S. Pat. No. 4,650,764;Temin et al., U.S. Pat. No. 4,980,289; Markowitz et al., 1988, J. Virol.62:1120 (1988); Temin et al., U.S. Pat. No. 5,124,263; Dougherty et al.,WO 95/07358; and Kuo et al., 1993, Blood 82:845. Targeted gene deliveryis described in WO 95/28494.

[0275] Alternatively, the vector can be introduced by lipofection. Forthe past decade, there has been increasing use of liposomes forencapsulation and transfection of nucleic acids in vitro. Syntheticcationic lipids designed to limit the difficulties and dangersencountered with liposome mediated transfection can be used to prepareliposomes for in vivo transfection of a gene encoding a marker (Felgneret. al., 1987, Proc. Natl. Acad. Sci. USA 84:7413-7417; Mackey et al.,1988, Proc. Natl. Acad. Sci. USA 85:8027-8031). The use of cationiclipids may promote encapsulation of negatively charged nucleic acids,and also promote fusion with negatively charged cell membranes (Felgner& Ringold, 1989, Science 337:387-388). The use of lipofection tointroduce exogenous genes into the specific organs in vivo has certainpractical advantages. Molecular targeting of liposomes to specific cellsrepresents one area of benefit. It is clear that directing transfectionto particular cell types would be particularly advantageous in a tissuewith cellular heterogeneity, such as pancreas, liver, kidney, and thebrain. Lipids may be chemically coupled to other molecules for thepurpose of targeting (see Mackey et al., 1988, supra). Targetedpeptides, e.g., hormones or neurotransmitters, and proteins such asantibodies, or non-peptide molecules could be coupled to liposomeschemically.

[0276] It is also possible to introduce the vector as a naked DNAplasmid. Naked DNA vectors for gene therapy can be introduced into thedesired host cells by methods known in the art, e.g., transfection,electroporation, microinjection, transduction, cell fusion, DEAEdextran, calcium phosphate precipitation, use of a gene gun, or use of aDNA vector transporter (see, e.g., Wu et al., 1992, J. Biol. Chem.267:963-967; Wu & Wu, 1988, J. Biol. Chem., 263:14621-14624; CanadianPatent Application No.2,012,311).

[0277] Naked nucleic acids encoding the active compound may also beintroduced using the gene-activated matrices described, for example, inU.S. Pat. No. 5,962,427.

[0278] Effective Dosages

[0279] The active compound(s) of the invention, or compositions thereof,will generally be used in an amount effective to treat or prevent theparticular disease being treated. The compound(s) may be administeredtherapeutically to achieve therapeutic benefit or prophylactically toachieve prophylactic benefit. By therapeutic benefit is meanteradication or amelioration of the underlying disorder being treated,e.g., eradication or amelioration of the underlying allergy, atopicdermatitis, atopic eczema or atopic asthma, and/or eradication oramelioration of one or more of the symptoms associated with theunderlying disorder such that the patient reports an improvement infeeling or condition, notwithstanding that the patient may still beafflicted with the underlying disorder. For example, administration ofan active compound to a patient suffering from an allergy providestherapeutic benefit not only when the underlying allergic response iseradicated or ameliorated, but also when the patient reports a decreasein the severity or duration of the symptoms associated with the allergyfollowing exposure to the allergen. Therapeutic benefit also includeshalting or slowing the progression of the disease, regardless of whetherimprovement is realized.

[0280] For prophylactic administration, the active compound may beadministered to a patient at risk of developing a disorder characterizedby, caused by or associated with IgE production and/or accumulation,such as the various disorders previously described. For example, if itis unknown whether a patient is allergic to a particular drug, theactive compound may be administered prior to administration of the drugto avoid or ameliorate an allergic response to the drug. Alternatively,prophylactic administration may be applied to avoid the onset ofsymptoms in a patient diagnosed with the underlying disorder. Forexample, an active compound may be administered to an allergy suffererprior to expected exposure to the allergen. Active compounds may also beadministered prophylactically to healthy individuals who are repeatedlyexposed to agents known to induce an IgE-related malady to prevent theonset of the disorder. For example, an active compound may beadministered to a healthy individual who is repeatedly exposed to anallergen known to induce allergies, such as latex allergy, in an effortto prevent the individual from developing an allergy.

[0281] The amount of active compound(s) administered will depend upon avariety of factors, including, for example, the particular indicationbeing treated, the mode of administration, whether the desired benefitis prophylactic or therapeutic, the severity of the indication beingtreated and the age and weight of the patient, the bioavailability ofthe particular active compound, etc. Determination of an effectivedosage is well within the capabilities of those skilled in the art.

[0282] Initial dosages may be estimated initially from in vitro assays.For example, an initial dosage for use in animals may be formulated toachieve a circulating blood or serum concentration of active compoundthat inhibits about 25% or more of IL-4 induced IgE production, or aprocess associated therewith, such as germline ε transcription, asmeasured in an in vitro assay. Alternatively, an initial dosage for usein animals may be formulated to achieve a circulating blood or serumconcentration of active compound that is equal to or greater than theIC₅₀ as measured in a CLLD8 protein competitive binding assay with anactive CL02A3 compound of the invention, such as peptide CL02A3 wt,peptide CL02A3LG or peptide CL02A3GW. Calculating dosages to achievesuch circulating blood or serum concentrations taking into account thebioavailability of the particular active compound is well within thecapabilities of skilled artisans. For guidance, the reader is referredto Fingl & Woodbury, “General Principles,” In: The Pharmaceutical Basisof Therapeutics, Chapter 1, pp. 1-46, 1975, and the references citedtherein.

[0283] Initial dosages can also be estimated from in vivo data, such asanimal models. Animals models useful for testing the efficacy ofcompounds to treat or prevent diseases characterized by, caused by orassociated with IgE production and/or accumulation are well-known in theart. Suitable animal models of hypersensitivity or allergic reactionsare described in Foster, 1995, Allergy 50(21Suppl):6-9, discussion 34-38and Tumas et al., 2001, J. Allergy Clin. Immunol. 107(6):1025-1033.Suitable animal models of allergic rhinitis are described in Szelenyi etal., 2000, Arzneimittelforschung 50(11):1037-42; Kawaguchi et al., 1994,Clin. Exp. Allergy 24(3):238-244 and Sugimoto et al., 2000,Immunopharmacology 48(1):1-7. Suitable animal models of allergicconjunctivitis are described in Carreras et al., 1993, Br. J.Ophthalmol. 77(8):509-514; Saiga et al., 1992, Ophthalmic Res.24(1):45-50; and Kunert et al., 2001, Invest. Ophthalmol. Vis. Sci.42(11):2483-2489. Suitable animal modules of systemic mastocytosis aredescribed in O'Keefe et al., 1987, J. Vet. Intern. Med. 1(2):75-80 andBean-Knudsen et al., 1989, Vet. Pathol. 26(1):90-92. Suitable animalmodels of hyper IgE syndrome are described in Claman et al., 1990, Clin.Immunol. Immunopathol. 56(1):46-53. Suitable animal models of B-celllymphoma are described in Hough et al., 1998, Proc. Natl. Acad. Sci. USA95:13853-13858 and Hakim et al., 1996, J. Immunol. 157(12):5503-5511.Suitable animal models of atopic disorders such as atopic dermatitis,atopic eczema and atopic asthma are described in Chan et al., 2001, J.Invest. Dermatol. 117(4):977-983 and Suto et al., 1999, Int. Arch.Allergy Immunol. 120(Suppl 1):70-75. Ordinarily skilled artisans canroutinely adapt such information to determine dosages suitable for humanadministration.

[0284] Dosage amounts will typically be in the range of from about 1mg/kg/day to about 100 mg/kg/day, 200 mg/kg/day, 300 mg/kg/day, 400mg/kg/day or 500 mg/kg/day, but may be higher or lower, depending upon,among other factors, the activity of the active compound, itsbioavailability, the mode of administration and various factorsdiscussed above. Dosage amount and interval may be adjusted individuallyto provide plasma levels of the active compound(s) which are sufficientto maintain therapeutic or prophylactic effect. In cases of localadministration or selective uptake, such as local topicaladministration, the effective local concentration of active compound(s)may not be related to plasma concentration. Skilled artisans will beable to optimize effective local dosages without undue experimentation.

[0285] The compound(s) may be administered once per day, a few orseveral times per day, or even multiple times per day, depending upon,among other things, the indication being treated and the judgement ofthe prescribing physician.

[0286] Preferably, the active compound(s) will provide therapeutic orprophylactic benefit without causing substantial toxicity. Toxicity ofthe active compound(s) may be determined using standard pharmaceuticalprocedures. The dose ratio between toxic and therapeutic (orprophylactic) effect is the therapeutic index. Active compound(s) thatexhibit high therapeutic indices are preferred.

[0287] The invention having been described, the following examples areoffered by way of illustration and not limitation.

EXAMPLES

[0288] Identification of Peptide CL02A3 wt from a Random Library ofPeptide 20-mers

[0289] Peptide CL02A3 wt was identified by screening a retrovirallibrary of random peptide 20-mers for the ability to inhibit IL-4induced germline ε transcription using the HBEGF2a/diphtheria dualreporter phenotypic screening system described in WO 01/31232. Toconstruct the random library, A5T4 reporter cells (described in moredetail below) were infected with an infectious retroviral library ofrandom peptide 20-mers (prepared as described in WO 97/27213; see alsoWO 01/34806 at page 39, line 36 through page 40, line 19). Theretroviral vector used includes a gene encoding blue fluorescent protein(BFP) fused upstream of the region encoding the random peptide via alinker region encoding an α-helical peptide linker (expression fusionproduct is referred to as “BFP-peptide”). Expression of the BFP-peptideproduct is controlled by a promoter sensitive to thetetracycline-regulated transactivator such that expression of theBFP-peptide is regulated by tetracycline (Tet) or doxycycline (Dox). SeeU.S. patent application Ser. No. 10/096,339, entitled “Methods andCompositions for Screening for Altered Cellular Phenotypes,” filed onMar. 8, 2002. The BFP reporter gene provides a rapid phenotypic assay todetermine whether cells were infected: infected cells expressBFP-peptide and fluoresce blue (phenotype BFP+), uninfected cells do notexpress BFP-peptide, and do not fluoresce blue (phenotype BFP-). Toreduce the number of stop codons, the region of the vector encoding therandom peptide was of the sequence (NNK)₂₀, where each N independentlyrepresents A, T, C or G and K represents T or G. The library was alsobiased to account for degeneracy in the genetic code.

[0290] The A5T4 reporter cell line was engineered from BJAB B-cells(Menezes et al., 1975, Biomedicine 22:276-284; Source: Yoshinobu Matsuo,PhD., Fujisaki Cell Center, Hayashibara Biochemical Labs, Inc., 675-1Fujisaki, Okayama 702-8006, Japan) and includes a reporter gene encodingthe HBEGF2a/GFP dual function reporter positioned downstream of anengineered 600 base pair IL-4 responsive fragment of an ε promoter (FIG.1; see also WO 99/58663) such that ultimate expression of the dualfunction reporter is driven by the ε promoter. When expressed, the dualfunction reporter cleaves into two pieces, a heparin-binding epidermalgrowth factor-like growth factor (HBEGF) and a green fluorescent protein(GFP), via the self-cleaving 2a sequence (Donnelly et al., 2001, J. Gen.Viol. 82:1027-1041; Donnelly et al., 1997, J. Gen. Virol. 78:13-21;Mattion et al., 1996, J. Virol. 70:8124; Ryan et al., 1994, EMBO J.13:928-33 Ryan et al., 1991, J. Gen. Virol. 72:2727; Hellen et al.,1989, Biochem. 28:9881; see also, WO 99/58663). In this reporter system,cells ectopically expressing HBEGF are capable of translocatingdiphtheria toxin (DT) into their cytoplasm, leading to rapid, acutecytotoxicity. Cells that do not express HBEGF are spared this fate andcontinue to survive even in the presence of high concentrations of DT.The A5T4 reporter cell line was further engineered to express thetetracycline-regulated transactivator (tTA), allowing for regulation ofpeptide library expression with tetracycline (Tet) or doxycycline (Dox).See U.S. patent application Ser. No. 10/096,339, entitled “Methods andCompositions for Screening for Altered Cellular Phenotypes,” filed onMar. 8, 2002, the disclosure of which is incorporated herein byreference. Thus, according to this dual phenotypic reporter system,unstimulated control cells expressing a random peptide fluoresce blue(BFP⁺) in the absence of Tet or Dox. In the presence of Tet or Dox, thepeptide is not made and the cells are BFP⁻. Following stimulation withIL-4, BFP⁺ cells expressing a non-inhibitory peptide fluoresce greenand, in addition are sensitive to DT. Stimulated BFP⁺ cells expressingan inhibitory peptide do not fluoresce green and are not DT sensitive.The toxin-conditional selection and Tet or Dox-controlled peptideexpression features of the A5T4 screening line are illustrated in FIGS.2A & 2B, respectively.

[0291] Following infection, the library was enriched for cellsexpressing peptides that inhibit IL-4 induced ε transcription asgenerally outlined in the top half of FIG. 3 and sorted by FACS intosingle cell clones. The clones were then screened as generallyillustrated in the lower half of FIG. 3. Briefly, for screening, eachclone was divided into two populations and one population was treatedwith Dox (10 ng/ml). After 5 days, both populations were stimulated withIL-4 (final conc. 60 U/mL; PeptroTech, Inc.) and, after 3 more days,both populations were analyzed by FACS to measure BFP and GFPfluorescence. FACS data were converted to a “reporter ratio”, which isdefined for this purpose as the ratio of the geometric mean of GFPfluorescence of the +IL-4/+Dox and +IL-4/-Dox populations. Cellsexpressing a peptide that inhibits germline ε transcription have areporter ratio of ≧1.1. A reporter ratio of ≧1.2 is indicative of stronginhibition.

[0292] The sequences of peptides expressed by positive clones (reporterratios of >1.1) were obtained by RT-PCR amplification of the integratedpeptide-expressing sequences. In this experiment, of 2.4×10⁹ A5T4 cellsinfected, 218 positive clones were identified, 199 of which were unique.From this same experiment, 155 total clones with a reporter ratioof >1.19 were identified, 136 of which were unique. Clone CL02A3, whichencodes peptide CL02A3 wt, was amongst the positive clones identified(clone CL02A3 had a reporter ratio of 1.55).

[0293] Clone CL02A3 Transfers Its Phenotype into Naïve Cells

[0294] The ability of peptide CL02A3 wt to inhibit germline εtranscription was confirmed in naive cells. Briefly, Phoenix cells weretransfected with a retroviral vector encoding a BFP-CL02A3 wt peptidefusion as described in WO 99/58663 and WO 97/27213. Naive A5T4 cellswere infected with the resultant virions and grown for 3 days. Theinfected cells were stimulated with IL-4 (60 U/mL) and, after 3 days,the cells were assessed by FACS for BFP and GFP. The FACS data arepresented in FIG. 4. As illustrated in FIG. 4, there are two populationsof cells: infected cells that express the BFP-peptide fusion (BFP⁺) anduninfected cells that do not (BFP⁻). The BFP fluorescence datacorresponding to the BFP⁺ and BFP⁻ populations are provided in Panel A.The GFP fluorescence data corresponding to the BFP⁺ and BFP⁻ populationsare presented in Panel B. The reporter ratio for this purpose isdetermined as the geometric mean of the GFP fluorescence of the BFP⁻population divided by the geometric mean of the GFP fluorescence of theBFP⁺ population. The reporter ratio for the CL02A3 in this re-infectionassay was 1.62.

[0295] Peptide CL02A3 wt Inhibits Transcription of an EndogenousGermline ε Promoter

[0296] The ability of peptide CL02A3 wt to inhibit transcription of anendogenous germline ε promoter was confirmed using a TAQMAN® assay(Roche Molecular, Alameda, Calif.). Briefly, A5T4 cells were infectedwith retrovirus capable of expressing peptide CL02A3 wt (prepared asdescribed above). The cells were sorted for BFP to select for infectedcells. Infected cells were divided into two populations. One populationwas exposed to Dox (10 ng/ml). Both populations were stimulated withIL-4 (60 U/ml). After 3 days, the cells were pelleted and the pelletsassayed for ε promoter transcription using a TAQMAN assay performed asdescribed in Applied Biosystems Protocol 4310299 (available athttp:\\www.appliedbiosystems.com). The primers and probe, which arespecific for the transcription product driven by the A5T4 endogenous εpromoter, were as follows (the probe was labeled at the 5′-end with Famand at the 3′-end with Tamra): ε forward primer: ATCCACAGGCACCAAATGGA(SEQ ID NO:11) ε reverse primer: GGAAGACGGATGGGCTCTG (SEQ ID NO:12)ε probe: ACCCGGCGCTTCAGCCTCCA (SEQ ID NO:13)

[0297] The measured endogenous ε inhibition ratio, defined as the ratioof the relative expression units (TAQMAN quantitative PCR of εtranscription product) of +IL-4/+Dox to +IL-4/-Dox cells, was 2.82(average of 3 values; p=0.0020), indicating that peptide CL02A3 wtstrongly inhibits the endogenous germline ε promoter.

[0298] Peptide CL02A3 wt is Selective for the Germline ε Promoter

[0299] To demonstrate selectivity for the germline E promoter, peptideCL02A3 wt was tested for inhibition of germline ε transcription. Theassay was similar to that described in the immediately precedingsection, except that ST486 cells (ATCC # CRL-1647) engineered to expressthe tetracycline-regulated transactivator were infected and the infectedcells were stimulated with TGF-β (40 ng/ml; Peprotech). The primers andprobe, which are specific for the transcription product driven by theST486 endogenous α promoter, were as follows (the probe was labeled atthe 5′-end with Fam and at the 3′-end with Tamra): α forward primer:CAGCACTGCGGGCCC (SEQ ID NO:14) α reverse primer: TCAGCGGGAAGACCTTGG (SEQID NO:15) α probe: CCAGCAGCCTGACCAGCATCCC (SEQ ID NO:16)

[0300] The measured endogenous α inhibition ratio was 0.89 (average of 3values; p=0.5430), indicating that peptide CL02A3 wt does not inhibittranscription of the germline α promoter. These data confirm thatpeptide CL02A3 wt is a selective inhibitor of germline ε transcription.

[0301] IgE Synthesis and ELISA Assay

[0302] This example describes an IgE synthesis mixed lymphocyte andELISA assay that may be used to assess the amount of IgE produced bycells such as human peripheral blood lymphocytes or other lymphaticcells in the presence and absence of candidate compounds, such as CL02A3compounds and/or compounds identified in the screening assays of theinvention.

[0303] IgE Synthesis Assay

[0304] (a) Materials

[0305] In the various protocols that follow below, the followingmaterials are used:

[0306] Heparin (Sigma H3393, St. Louis, Mo.)

[0307] Histopaque 1077 tubes (Sigma A0561, St. Louis, Mo.)

[0308] Iscove's Modified Dulbeccos Medium (“IMDM); Sigma 13390, St.Louis, Mo.)

[0309] Bovine Serum Albumin (“BSA”; Sigma A9418, St. Louis, Mo.).

[0310] Fetal Bovine Serum (“FCS”; Sigma F7524, St. Louis, Mo.). (theserum is heat inactivated prior to use at 56° C. for 30 minutes,aliquoted and stored at −20° C.; “HI-FCS”) Human Transferrin (SigmaT2252, St. Louis, Mo.)

[0311] Bovine Insulin (Sigma 11882, St. Louis, Mo.)

[0312] 200 mM L-Glutamine (“L-Gln”; Sigma G7513, St. Louis, Mo.) (storedas 5 ml aliquots at −20° C.)

[0313] Pen/Strep 10% solution (Sigma P0781, St. Louis, Mo.) (stored as 5ml aliquots at −20° C.)

[0314] PBS Dulbeccos (Gibco BRL 14190-094, now Invitrogen, Carlsbad,Calif.)

[0315] DMSO (Sigma D2650, St. Louis, Mo.)

[0316] Recombinant Human IL-4 (R&D Systems 204-IL, Minneapolis, Minn.)(stored as a stock solution of 100,000 U/ml in culture medium at −20°C.)

[0317] 96 well tissue culture plates (Costar 3595, Coming Inc., LifeSciences, Acton MA)

[0318] 96 well dilution blocks (Porvair 219008, Shepperton, UK)

[0319] Culture Medium: supplement 500 ml IMDM with 0.5% BSA (2.5 g), 10%HI-FCS (50 ml), 25 mg human transferrin, 2.5 mg bovine insulin, 2 mML-Gln (5 ml) and 1% pen/strep (5 mL). Filter sterilize before use.

[0320] (b) Blood Collection

[0321] Make up a 1 mg/ml solution of heparin in sterile PBS and place 1ml in each sterile 50 ml centrifuge tube required. Collect 50 ml ofvenous blood from a healthy human volunteer per centrifuge tube.

[0322] (c) Lymphocyte Isolation

[0323] Lymphocytes are isolated from the blood according to the protocolbelow:

[0324] 1. Dilute blood with an equal volume of PBS containing 2% HI-FCS.

[0325] 2. Add 20 ml diluted blood to each histopaque tube. Thehistopaque tubes should be warmed to room temperature before use. Theycan be left overnight at room temp in the dark and then spun at 1000 rpmfor 5 min to settle the contents before use. Spin at 1000 g for 35 minat room temperature in a benchtop centrifuge with the break set to off.

[0326] 3. Draw off the upper plasma layer and discard.

[0327] 4. Draw off the lymphocyte layer into a sterile centrifuge tube.If there is clear definition between the bottom of the lymphocyte layerand the top of the frit, remove only the lymphocyte layer, not all theliquid above the frit.

[0328] 5. Add 30 ml PBS-2% HI-FCS to each 10 ml of cell suspension andspin at 1000 rpm for 10 min at room temp.

[0329] 6. Discard the supernatant and resuspend each cell pellet in 5-10ml PBS-2%HI-FCS. Transfer the suspensions to a single tube and bring thevolume to 40 ml with PBS-2% HI-FCS. Spin at 1000 rpm for 10 min at roomtemp.

[0330] 7. Repeat Step 6.

[0331] 8. Wash the cells once with 40 ml culture medium.

[0332] 9. Discard the supernatant and resuspend the pellet in 10 ml orless culture medium.

[0333] 10. Count the cells (using a Neubauer haemocytometer, a CoulterMax-M cell counter or other counter).

[0334] 11. Resuspend the cells in culture medium to a concentration of2×10⁶ cells/ml. The cells can be left at this stage until they areneeded for assay set up.

[0335] (d) Assay Set Up

[0336] The assay is carried out as follows:

[0337] 1. Dissolve test compounds in DMSO to give a 10 mM stocksolution. If necessary, sonicate the stock solution to aid dissolutionof the compound.

[0338] 2. Dilute each compound stock solution 1:20 with culture mediumto yield a 500 μM solution in 5% DMSO. Serially dilute this 500 μM stocksolution 1:10 with culture medium several times to provide enough stocksolutions to test the compounds over a range of concentrations (e.g.,from 1 nM to 10 μM).

[0339] 3. Just prior to use, prepare a stock solution of IL-4 (1000 U/mlin culture medium).

[0340] 4. To test the compounds and prepare appropriate controls, add tothe appropriate wells of a multiwell plate the following reagents in thefollowing amounts: Reagent + IL-4 control − IL-4 control Sample TestCompound − − 50 μl IL-4 50 μl − 50 μl Culture Medium − 50 μl − 0.5% DMSOin 50 p1 50 μl − Culture Medium Cells 150 μl  150 μl  150 μl  TotalVolume 250 μl  250 μl  250 μl 

[0341] 5. Incubate the plates for 10-12 days at 37° C. in a CO₂incubator (5% CO₂/95% O₂). Following incubation, spin the plates at 1000rpm for 10 min and store at −20° C. until ready for the ELISA detectionassay that follows below.

[0342] ELISA Assay for Detection of IgE

[0343] (a) Materials

[0344] In the assay protocol that follows below, the following materialsare used:

[0345] Nunc maxisorp ELISA plates (GIBCO Brl, now Invitrogen, Carlsbad,Calif.) Murine anti human IgE (GE1 clone) (Sigma, St. Louis, Mo.)

[0346] Phosphate buffered saline (“PBS”; Sigma. St. Louis, Mo.)

[0347] Bovine serum albumin (“BSA”; Sigma, St. Louis, Mo.)

[0348] Polyethylenesorbitan monolaurate (“TWEEN 20”; Sigma, St. Louis,Mo.)

[0349] OPD tablets (DAKO Corp., Carpenteria, Calif.)

[0350] Streptavidin biotinylated horseradish peroxidase complex(“Streptavidin HRP”; Amersham, Piscataway, N.J.)

[0351] Human Myeloma protein IgE (The Binding Site, Inc.; San Diego,Calif.)

[0352] Biotinylated anti human IgE (Vector Laboratories, Inc.,Burlingame, Calif.) Hydrogen peroxide (Sigma, St. Louis, Mo.)

[0353] Distilled water (“dH₂O”)

[0354] Buffer A: 0.1 M NaHCO₃, pH 9.6

[0355] Buffer B: 0.1% TWEEN 20 in PBS

[0356] Buffer C: 1% BSA in Buffer B

[0357] Stop Solution: 0.6M H₂SO₄

[0358] (a) Protocol

[0359] The assay which measures the amount of IgE synthesized by thevarious controls and samples prepared above, is carried out as describedin the following protocol:

[0360] 1) Coat Nunc maxisorp plates with 50 μl of murine anti human IgE(1:2000 in Buffer A). Leave plates at 4° C. overnight. Plates can bestored in this way for a maximum of 7 days.

[0361] 2) Wash plates 3× with Buffer.

[0362] 3) Block any unbound sites on the plate by adding 200 μl ofbuffer C. Incubate the plates for at least 2 hours at room temperature.If blocking overnight incubate at 4 C. Blocked plates should only bekept for a maximum of 2 days.

[0363] 4) Wash plates 3×with Buffer B.

[0364] 5) Place 50 μl of the sample or standard IgE diluted in Buffer Cto each well. IgE standards are diluted to give a concentration range of100 ng/ml-0 ng/ml. To make up the 100 ng/ml standard, carry out thefollowing dilutions of stock IgE (0.5 mg/ml) in buffer C: 1 in 50 togive a 10 μg/ml solution (a minimum of 10 μs must be transferred fromthe stock) followed by a 1 in 10 to give 1 μg/ml and then a further 1 in10 to give 100 ng/ml. Double dilutions are then carried out in buffer Cto give the rest of the standard curve. Carry out all dilutions in glassbottles (10 oz) and make up at least 1 ml of each so that reasonablevolumes are being transferred (one ELISA plate requires 100 μls of eachstandard). For the pilot ELISA the standard curve is added to each platejust after the samples have been added. Set up one ELISA plate with 3-4columns of standards only. This will be used to determine developmenttime at protocol Step 11.

[0365] For the pilot ELISA incubate the plates for one-two hours at roomtemperature

[0366] For the full ELISA incubate the plates overnight at 4° C.

[0367] 6) Wash plates 3× with Buffer B.

[0368] 7) Add 50 μl of biotinylated anti human IgE (1/500 dilution inBuffer B) to all wells. Incubate the plate for 1 hour at roomtemperature.

[0369] 8) Wash plates 3×with Buffer B

[0370] 9), Add 50 μl of streptavidin HRP to each well (1/800 dilution inBuffer B). Incubate the plates for 45 mins to 1 hour at roomtemperature. The plates must not be left for longer than 1 hour at thisstage.

[0371] 10) Wash plates 3×with Buffer B.

[0372] 11) Add 50 μl of substrate (4 OPD tablets per 12 ml dH₂O plus 5μl hydrogen peroxide per 12 ml) to each well and wait for the color todevelop (this usually occurs within 10 minutes). Determine the timetaken to give the required OD (1.5-2 for 100 ng/ml of standard IgE)using a plate containing only a standard curve. Develop all test platesfor this amount of time and in batches of 5 plates. This is especiallyimportant if there are a large number of plates.

[0373] 12) Quench the reaction by adding 50 μl Stop Solution.

[0374] 13) Read plates at 492 nm within 30 minutes of stopping thereaction.

[0375] Peptide CL02A3 wt Mediates its Inhibitory Action by Binding CLLD8protein Identification of Potential Binding Partners for Peptide CL02A3wt

[0376] Potential binding partners for peptide CL02A3 wt were identifiedin a β-galactosidase yeast two-hybrid (YTH) assay using peptide CL02A3wt as bait and a cDNA library constructed from the A5T4 reporter cellline as prey. Binding was assessed by β-galactosidase quantificationusing BetaFluor (Novagen) as a substrate. A negative interaction control(no cDNA fused downstream of the GAL4 activation domain sequence) wasalso run. A general outline of the YTH assay is illustrated in FIG. 5A.Following clustering, filtering to remove non-specific bait hits (e.g.,GFP and BFP), singletons and clusters recognized by 10 or more cDNAbaits (based upon historical YTH assays), and prioritization, 64 preyclones were identified as hits.

[0377] Potential targets identified in the YTH assay were reconfirmed asgenerally outlined in FIG. 6. The cDNA clones identified as hits in theYTH assay were purified and rescreened for interaction with the CL02A3wt peptide in a YTH assay. The hit clones were also screened in acontrol assay for interaction with the BFP using a vector (pGBKT7) thatcontained only the BFP and not the CL02A3 peptide. At this stage, 51putative target clones remained.

[0378] In order to further discriminate among the putative targets,functional/interaction profile analyses were carried out as described incopending application Ser. No. 10/095,659, entitled Methods ofIdentifying Protein Targets, filed Mar. 8, 2002, attorney docketRIGL-003/00US.

[0379] Confirmation that Peptide CL02A3 wt Mediates Its InhibitoryAction by Binding CLLD8 Protein

[0380] Confirmation of colony 4.2CL02A3_Y_GB_UN_(—)0739 as the bindingpartner for Peptide CL02A3 wt was confirmed using the profiling methoddescribed in copending application Ser. No. 10/095,659, entitled Methodsof Identifying Protein Targets, filed Mar. 8, 2002 (identified byattorney docket no. RIGL-003/00US), the entire disclosure of which isincorporated herein by reference. The functional profile was obtainedusing the A5T4 reporter cell line and the interaction profiles wereobtained using the YTH assay and compared for correspondence. The mainconcept underlying this profiling method is that mutants will tend toact the same way in both the functional assay and an interaction assaywith the target polypeptide of the wild-type peptide. That is, a mutantthat exhibits an increase in function (as compared to the wild-typepeptide) in the functional assay will exhibit an increase in interaction(as compared to the wild-type peptide) in a YTH assay with the targetpolypeptide of the wild-type peptide. Stated another way, the targetpolypeptide will yield an interaction profile that corresponds closelyto the functional profile when compared visually or by other means.1002191 A collection of CL02A3 mutants was generated by replacing singleor double amino acid residues in the sequence of CL02A3 wt with adifferent amino acid, typically an alanine or glycine, as described incopending application Ser. No. 10/095,659, entitled Methods ofIdentifying Protein Targets, filed Mar. 8, 2002 attorney docketRIGL-003/00US. The functional profiles for the mutants derived fromCL02A3 wt was obtained by constructing and screening for activity in theA5T4 reporter cell line in the manner described above. The activity ofeach mutant at the germline ε promoter is reflected in the reporterratio, as described above. The reporter ratios were the functionalvalues that were used to develop the functional profiles.

[0381] TABLE 2 depicts the amino acid sequences (the dual alaninemutations are underlined) of the mutants tested and the measuredreporter ratios when screening in the A5T4 reporter line. TABLE 2 Re-Peptide porter Name Peptide Sequence Ratio SEQ ID NO CL02A3wtAMHGHHGWPWGMEQGCTPLG 1.62 (SEQ ID NO:1) CL02A3LG AMHGHHGWPWGMEQGCTPAA1.99 (SEQ ID NO:2) CL02A3GW AMHGHHAAPWGMEQGCTPLG 1.16 (SEQ ID NO:3)CL02A3TP AMHGHHGWPWGMEQGCAALG 1.91 (SEQ ID NO:4) CL02A3EQAMHGHHGWPWGMAAGCTPLG 2.23, (SEQ ID NO:5) CL02A3HG AMAAHHGWPWGMEQGCTPLG1.5 (SEQ ID NO:6) CL02A3GC AMHGHHGWPWGMEQAATPLG 1.89 (SEQ ID NO:7)CL02A3HH AMHGAAGWPWGMEQGCTPLG 1.29 (SEQ ID NO:8) CL02A3PWAMHGHHGWAAGMEQGCTPLG 1.24 (SEQ ID NO:9) CL02A3AM RAHGHHGWPWGMEQGCTPLG1.03 (SEQ ID NO:10)

[0382] The interaction of CL02A3 wt peptide and its mutants with theclones identified as potential targets for the peptide CL02A3 wt wasthen quantified in a β-galactosidase YTH assay. The YTH assay wasperformed in the manner described above. A general outline of this YTHassay is illustrated in FIG. 5B. For each clone tested, an interactionprofile was developed by comparing the β-galactosidase activity of eachmutant to that of the wild type peptide, as described above.

[0383] Comparison of the interaction and functional profiles wasperformed by categorizing the mutants of CL02A3 based on the functionaland interaction assays. Based on the reporter ratio, described in panelC of FIG. 4, each mutant was categorized into one of four functionalcategories: (1) reduction of function (ROF); (2) loss of function (LOF);(3) increase of function (IOF) or (4) functionally neutral (N), ascompared to the activity of the wild type peptide, here CL02A3 wt. Asmentioned previously, cells expressing a peptide that inhibits germlineε transcription have reporter ratios of ≧1.1. Cells expressing a loss offunction mutant have a reporter ratio of <1.11. An increase of functionmutant (IOF) shows a >50% increase in reporter ratio and a reduction offunction of mutant shows a >50% decrease in reporter ratio. Functionallyneutral mutants have reporter ratios that fall within ±50% of that ofthe CL02A3 wt peptide. The % increase or decrease in reporter ratio iscalculated after subtracting 1.0 from the individual ratios. Using thesecriteria, peptides CL02A3LG (SEQ ID NO:2), CL02A3TP (SEQ ID NO:4), andCL02A3EQ (SEQ ID NO:5) were designated as IOF mutants, peptide CL02A3LGW(SEQ ID NO:3), CL02A3HH (SEQ ID NO:8), CL02A3PW (SEQ ID NO:9) weredesignated as ROF mutants, peptides CL02A3HG (SEQ ID NO:6), and CL02A3GC(SEQ ID NO:7) were designated as neutral, and peptide CL02A3AM (SEQ IDNO: 10) was designated as a LOF mutant.

[0384] The interaction of these IOF, ROF and LOF mutants with differentpolypeptides encoded by the putative target clones' were quantifiedusing the YTH assay described above. Based on the YTH assay, for eachputative target, a subset of the mutants representing each of the abovefunctional categories, when available, were categorized into thefollowing four interaction categories: (1) reduction of interaction(ROI); (2) loss of interaction (LOI); (3) increase of interaction (101);and (4) interactionally neutral (N), by assessing the binding affinityratio of the mutant peptide and the wild type peptide (β-gal activity ofmutant/wild type) for the putative target. For the YTH interactionassay, mutants CL02A3LG, CL02A3GW, and CL02A3AM, as well as theoriginally identified peptide CL02A3 wt were used. The selectioncriteria for categorizing the interactions is shown in FIG. 8.

[0385] Based on these functional and interaction categorizations,graphic profile representations for each potential target polypeptidewere obtained using a weighted categorization process as depicted inFIG. 9. In FIG. 9, the functional profile (reporter ratios of CL02A3 wtand three mutants) is plotted along the X-axis and the interactionprofile (β-galactosidase signal from the YTH assay for CL02A3 wt and itsthree mutants) is plotted along the Y-axis. The X-axis is furthercategorized into L (loss of function), R (reduction of function), N(functionally neutral), and I (increase of function) based on thereporter ratios, as described above. Similarly, the Y-axis is furthercategorized based on the criteria in FIG. 8. In such a graphic profile,profiles that correspond closely have interaction values (in this case,β-galactosidase signal) and functional values (in this case, reporterratios) that fall along a line with a positive slope. Also, closecorrespondence is indicated when the mutants fall within the samecategory type using both the reporter ratio and the β-galactosidasesignal, i.e., a mutant categorized as a L based on the reporter ratio isalso categorized as a L based on the β-galactosidase signal. In thiscase, the interaction and functional profiles for clone4.2CL02A3_Y_GB_UN_(—)0739 fall along a line with a positive slope. Eachof the three mutants also fall within the same respective categoriesbased on both reporter ratio and β-galactosidase signal. The profilesfor the other putative target clones selected in the initial YTH screendo not satisfy these criteria.

[0386] Based on the close correspondence observed between theinteraction and functional profiles, the polypeptide encoded by clone4.2CL02A3_Y_GB_UN_(—)0739 was identified as a target for peptide CL02A3wt. The clone was sequenced and a sequence comparison (using a Blastsearch with default parameters) with sequences in the NCBI (GenBank)protein database was carried out. From this sequence comparison clone4.2CL02A3_Y_GB_UN_(—)0739 was identified as encoding human CLLD8, NCBIaccession # NP_(—)114121 (also called CLLL8), nucleotide sequence atNCBI sequence database NM_(—)031915.

[0387] While the invention has been described by reference to variousspecific embodiments, skilled artisans will recognize that numerousmodifications may be made thereto without departing from the spirit andthe scope of the appended claims.

[0388] All references cited throughout the disclosure are incorporatedherein by reference in their entireties for all purposes.

1 18 1 20 PRT Artificial Sequence peptide encoded by synthetic oligomer1 Ala Met His Gly His His Gly Trp Pro Trp Gly Met Glu Gln Gly Cys 1 5 1015 Thr Pro Leu Gly 20 2 20 PRT Artificial Sequence peptide encoded bysynthetic oligomer 2 Ala Met His Gly His His Gly Trp Pro Trp Gly Met GluGln Gly Cys 1 5 10 15 Thr Pro Ala Ala 20 3 20 PRT Artificial Sequencepeptide encoded by synthetic oligomer 3 Ala Met His Gly His His Ala AlaPro Trp Gly Met Glu Gln Gly Cys 1 5 10 15 Thr Pro Leu Gly 20 4 20 PRTArtificial Sequence peptide encoded by synthetic oligomer 4 Ala Met HisGly His His Gly Trp Pro Trp Gly Met Glu Gln Gly Cys 1 5 10 15 Ala AlaLeu Gly 20 5 20 PRT Artificial Sequence peptide encoded by syntheticoligomer 5 Ala Met His Gly His His Gly Trp Pro Trp Gly Met Ala Ala GlyCys 1 5 10 15 Thr Pro Leu Gly 20 6 20 PRT Artificial Sequence peptideencoded by synthetic oligomer 6 Ala Met Ala Ala His His Gly Trp Pro TrpGly Met Glu Gln Gly Cys 1 5 10 15 Thr Pro Leu Gly 20 7 20 PRT ArtificialSequence peptide encoded by synthetic oligomer 7 Ala Met His Gly His HisGly Trp Pro Trp Gly Met Glu Gln Ala Ala 1 5 10 15 Thr Pro Leu Gly 20 820 PRT Artificial Sequence peptide encoded by synthetic oligomer 8 AlaMet His Gly Ala Ala Gly Trp Pro Trp Gly Met Glu Gln Gly Cys 1 5 10 15Thr Pro Leu Gly 20 9 20 PRT Artificial Sequence peptide encoded bysynthetic oligomer 9 Ala Met His Gly His His Gly Trp Ala Ala Gly Met GluGln Gly Cys 1 5 10 15 Thr Pro Leu Gly 20 10 20 PRT Artificial Sequencepeptide encoded by synthetic oligomer 10 Arg Ala His Gly His His Gly TrpPro Trp Gly Met Glu Gln Gly Cys 1 5 10 15 Thr Pro Leu Gly 20 11 20 DNAArtificial Sequence synthetic primer 11 atccacaggc accaaatgga 20 12 19DNA Artificial Sequence synthetic primer 12 ggaagacgga tgggctctg 19 1320 DNA Artificial Sequence synthetic primer 13 acccggcgct tcagcctcca 2014 15 DNA Artificial Sequence synthetic primer 14 cagcactgcg ggccc 15 1518 DNA Artificial Sequence synthetic primer 15 tcagcgggaa gaccttgg 18 1622 DNA Artificial Sequence synthetic primer 16 ccagcagcct gaccagcatc cc22 17 603 DNA Homo sapiens 17 ctcgaggaca gtgacctggg agtgagtacaaggtgaggcc accactcagg gtgccagctc 60 caagcgggtc acagggacga gggctgcggccatcaggagg ccctgcacac acatctggga 120 cacgcgcccc cgagggccag ttcacctcagtgcgcctcat tctcctgcac aaaagcgccc 180 ccatcctttc ttcacaaggc tttcgtggaagcagaggcgt cgatgcccag taccctctcc 240 ctttcccagg caacgggacc ccaagtttgctgactgggac caccaagcca cgcatgcgtc 300 aagagtgaga gtccgggacc taggcaggggccctggggtt gggcctgaga gagaagagaa 360 cctcccccag cactcggtgt gcatcggtagtgaaggagcc tcacctgacc cccgctgttg 420 ctcaatcgac ttcccaagaa cagagagaaaagggaacttc cagggcggcc cgggcctcct 480 gggggttccc accccatttt tagctgaaagcactgaggca gagctccccc tacccaggct 540 ccactgcccg gcacagaaat aacaaccacggttactgatc atctgggagc tgtccaggaa 600 ttc 603 18 719 PRT Homo sapiens 18Met Gly Glu Lys Asn Gly Asp Ala Lys Thr Phe Trp Met Glu Leu Glu 1 5 1015 Asp Asp Gly Lys Val Asp Phe Ile Phe Glu Gln Val Gln Asn Val Leu 20 2530 Gln Ser Leu Lys Gln Lys Ile Lys Asp Gly Ser Ala Thr Asn Lys Glu 35 4045 Tyr Ile Gln Ala Met Ile Leu Val Asn Glu Ala Thr Ile Ile Asn Ser 50 5560 Ser Thr Ser Ile Lys Gly Ala Ser Gln Lys Glu Val Asn Ala Gln Ser 65 7075 80 Ser Asp Pro Met Pro Val Thr Gln Lys Glu Gln Glu Asn Lys Ser Asn 8590 95 Ala Phe Pro Ser Thr Ser Cys Glu Asn Ser Phe Pro Glu Asp Cys Thr100 105 110 Phe Leu Thr Thr Gly Asn Lys Glu Ile Leu Ser Leu Glu Asp LysVal 115 120 125 Val Asp Phe Arg Glu Lys Asp Ser Ser Ser Asn Leu Ser TyrGln Ser 130 135 140 His Asp Cys Ser Gly Ala Cys Leu Met Lys Met Pro LeuAsn Leu Lys 145 150 155 160 Gly Glu Asn Pro Leu Gln Leu Pro Ile Lys CysHis Phe Gln Arg Arg 165 170 175 His Ala Lys Thr Asn Ser His Ser Ser AlaLeu His Val Ser Tyr Lys 180 185 190 Thr Pro Cys Gly Arg Ser Leu Arg AsnVal Glu Glu Val Phe Arg Tyr 195 200 205 Leu Leu Glu Thr Glu Cys Asn PheLeu Phe Thr Asp Asn Phe Ser Phe 210 215 220 Asn Thr Tyr Val Gln Leu AlaArg Asn Tyr Pro Lys Gln Lys Glu Val 225 230 235 240 Val Ser Asp Val AspIle Ser Asn Gly Val Glu Ser Val Pro Ile Ser 245 250 255 Phe Cys Asn GluIle Asp Ser Arg Lys Leu Pro Gln Phe Lys Tyr Arg 260 265 270 Lys Thr ValTrp Pro Arg Ala Tyr Asn Leu Thr Asn Phe Ser Ser Met 275 280 285 Phe ThrAsp Ser Cys Asp Cys Ser Glu Gly Cys Ile Asp Ile Thr Lys 290 295 300 CysAla Cys Leu Gln Leu Thr Ala Arg Asn Ala Lys Thr Ser Pro Leu 305 310 315320 Ser Ser Asp Lys Ile Thr Thr Gly Tyr Lys Tyr Lys Arg Leu Gln Arg 325330 335 Gln Ile Pro Thr Gly Ile Tyr Glu Cys Ser Leu Leu Cys Lys Cys Asn340 345 350 Arg Gln Leu Cys Gln Asn Arg Val Val Gln His Gly Pro Gln ValArg 355 360 365 Leu Gln Val Phe Lys Thr Glu Gln Lys Gly Trp Gly Val ArgCys Leu 370 375 380 Asp Asp Ile Asp Arg Gly Thr Phe Val Cys Ile Tyr SerGly Arg Leu 385 390 395 400 Leu Ser Arg Ala Asn Thr Glu Lys Ser Tyr GlyIle Asp Glu Asn Gly 405 410 415 Arg Asp Glu Asn Thr Met Lys Asn Ile PheSer Lys Lys Arg Lys Leu 420 425 430 Glu Val Ala Cys Ser Asp Cys Glu ValGlu Val Leu Pro Leu Gly Leu 435 440 445 Glu Thr His Pro Arg Thr Ala LysThr Glu Lys Cys Pro Pro Lys Phe 450 455 460 Ser Asn Asn Pro Lys Glu LeuThr Met Glu Thr Lys Tyr Asp Asn Ile 465 470 475 480 Ser Arg Ile Gln TyrHis Ser Val Ile Arg Asp Pro Glu Ser Lys Thr 485 490 495 Ala Ile Phe GlnHis Asn Gly Lys Lys Met Glu Phe Val Ser Ser Glu 500 505 510 Ser Val ThrPro Glu Asp Asn Asp Gly Phe Lys Pro Pro Arg Glu His 515 520 525 Leu AsnSer Lys Thr Lys Gly Ala Gln Lys Asp Ser Ser Ser Asn His 530 535 540 ValAsp Glu Phe Glu Asp Asn Leu Leu Ile Glu Ser Asp Val Ile Asp 545 550 555560 Ile Thr Lys Tyr Arg Glu Glu Thr Pro Pro Arg Ser Arg Cys Asn Gln 565570 575 Ala Thr Thr Leu Asp Asn Gln Asn Ile Lys Lys Ala Ile Glu Val Gln580 585 590 Ile Gln Lys Pro Gln Glu Gly Arg Ser Thr Ala Cys Gln Arg GlnGln 595 600 605 Val Phe Cys Asp Glu Glu Leu Leu Ser Glu Thr Lys Asn ThrSer Ser 610 615 620 Asp Ser Leu Thr Lys Phe Asn Lys Gly Asn Val Phe LeuLeu Asp Ala 625 630 635 640 Thr Lys Glu Gly Asn Val Gly Arg Phe Leu AsnHis Ser Cys Cys Pro 645 650 655 Asn Leu Leu Val Gln Asn Val Phe Val GluThr His Asn Arg Asn Phe 660 665 670 Pro Leu Val Ala Phe Phe Thr Asn ArgTyr Val Lys Ala Arg Thr Glu 675 680 685 Leu Thr Trp Asp Tyr Gly Tyr GluAla Gly Thr Val Pro Glu Lys Glu 690 695 700 Ile Phe Cys Gln Cys Gly ValAsn Lys Cys Arg Lys Lys Ile Leu 705 710 715

What is claimed is:
 1. A method of identifying a compound that modulatesIL-4 receptor-mediated IgE production or a process associated therewith,comprising determining whether the compound binds a CLLD8 protein,wherein the ability to bind the CLLD8 protein identifies the compound asbeing a modulator of IL-4 induced IgE production or a process associatedtherewith.
 2. The method of claim 1 in which the compound identifiedinhibits IL-4 receptor-mediated IgE production.
 3. The method of claim 1in which the compound identified inhibits IL-4 receptor-mediated isotypeswitching of B-cells.
 4. The method of claim 1 in which the compoundidentified inhibits IL-4 induced transcription of a germline ε promoter.5. The method of claim 1 in which the compound specifically modulatesIL-4 induced IgE production or a process associated therewith.
 6. Themethod of claim 1 in which the CLLD8 protein is a mammalian CLLD8protein.
 7. The method of claim 6 in which the mammalian CLLD8 proteinis a human CLLD8 protein.
 8. The method of claim 1 in which it isdetermined whether the compound binds the CLLD8 protein in a competitivebinding assay.
 9. The method of claim 8 in which the compound competesfor binding the CLLD8 protein with an active CL02A3 compound.
 10. Themethod of claim 9 in which the active CL02A3 compound is selected fromthe group consisting of CL02A3 wt (SEQ ID NO:1), CL02A3LG (SEQ ID NO:2),CL02A3GW (SEQ ID NO:3) and an analog thereof.
 11. The method of claim 1which is carried out in a cell-free system with an isolated CLLD8protein.
 12. The method of claim 1 in which the compound is a smallorganic compound.
 13. The method of claim 12 in which the small organiccompound has a molecular weight in the range of about 100-2500 dalton.14. The method of claim 1 in which the compound is selected from thegroup consisting of peptides and peptide analogs.
 15. The method ofclaim 1, further including the step of confirming that the identifiedcompound modulates IL-4 induced IgE production or a process associatedtherewith.
 16. A method of identifying compounds that modulate IL-4receptor-mediated IgE production or a process associated therewith,comprising the step of contacting a compound from pool of candidatecompounds with a CLLD8 protein and identifying those compounds of thepool that bind the CLLD8 protein, thereby identifying those compounds ofthe pool that modulate IL-4 receptor-mediated IgE production or aprocess associated therewith.
 17. The method of claim 16 which iscarried out in the presence of a compound known to bind the CLLD8protein such that those compounds of the pool that competitively bindthe CLLD8 protein are identified.
 18. The method of claim 17 in whichthe compound known to bind the CLLD8 protein is an active CL02A3compound.
 19. The method of claim 18 in which the active CL02A3 compoundis selected from the group consisting of CL02A3 wt (SEQ ID NO:1),CL02A3LG (SEQ ID NO:2), CL02A3GW (SEQ ID NO:3) and an analog thereof.20. The method of claim 16 in which the candidate compounds are peptidesor small organic compounds.
 21. The method of claim 20 in which the poolof candidate compounds is a phage display library.
 22. The method ofclaim 20 in which the candidate compounds are immobilized on a substrateor a plurality of substrates.
 23. The method of claim 16 in which thecompound or CLLD8 protein is labeled with a detectable label.
 24. Themethod of claim 16 in which the CLLD8 protein is immobilized on asubstrate.
 25. A compound comprising a peptide or peptide analog, or apharmaceutically acceptable salt thereof, having the formula (I):Z¹-X¹˜X²˜X⁴˜X⁵˜X⁶˜X⁷˜X⁸X⁹˜X¹⁰˜X¹¹X¹²˜X¹³˜Z¹⁴˜X¹⁵˜X¹⁶˜X¹⁷˜X¹⁹˜X¹⁹˜X²⁰-Z²wherein: X¹ is a small aliphatic residue; X² is a non-polar residue; X³is an aromatic residue, a basic residue or a Ala residue; X⁴ is a Gly orAla residue; X⁵ is an aromatic residue, a basic residue or a Alaresidue; X⁶ is an aromatic residue, a basic residue or a Ala residue; X⁷is an aliphatic residue; X⁸ is an aliphatic residue or an aromaticresidue; X⁹ is a conformationally-constrained residue or a Ala residue;X¹⁰ is an aromatic residue or a Ala residue; X¹¹ is a Gly or Alaresidue; X¹² is a non-polar residue; X¹³ is an acidic residue or a Alaresidue; X¹⁴ is a polar residue or a Ala residue; X¹⁵ is a Gly or Alaresidue; X¹⁶ is a cysteine-like residue or a Ala residue; X¹⁷ is ahydroxyl-containing residue or a Ala residue; X¹⁸ is a hydrophobicresidue; X¹⁹ is an aliphatic residue; X²⁰ is an aliphatic residue; Z¹ isRRN—, RC(O)NR—, RS(O)₂NR— or an amino-terminal blocking group; Z² is—C(O)OR, —C(O)O—, —C(O)NRR or a carboxyl-terminal blocking group; each Ris independently selected from the group consisting of hydrogen, alkyl,substituted alkyl, aryl, substituted aryl, arylalkyl, substitutedarylalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl andsubstituted heteroarylalkyl; each “˜” independently represents an amide,a substituted amide or an isostere of an amide; each “-” represents abond, or a 1 to 10 residue peptide or peptide analog; and wherein one ormore of X¹, X², X¹⁹, or X²⁰ may be absent.
 26. The compound of claim 25in which each “˜” is an amide, Z¹ is H₂N— and Z² is —C(O)OH or —C(O)O⁻.27. The compound of claim 25 having the formula:AMHGHHX⁷X⁸PWGMEQGCTPX¹⁹X²⁰ wherein: X⁷ is a small aliphatic residue; X⁸is an aromatic or a small aliphatic residue; X¹⁹ is a small aliphaticresidue; and X²⁰ is a small aliphatic residue.
 28. The compound of claim27 in which X⁷ is G or A and/or X⁸ is W or A.
 29. The compound of claim27 in which X¹⁹ is L or A and/or X²⁰ is A or G.
 30. The compound ofclaim 25 which is selected from the group consisting of CL02A3 wt (SEQID NO:1), CL02A3LG (SEQ ID NO:2), CL02A3GW (SEQ ID NO:3), CL02A3TP (SEQID NO:4), CL02A3EQ (SEQ ID NO:5), CL02A3HG (SEQ ID NO:6), CL02A3GC (SEQID NO:7), CL02A3HH (SEQ ID NO:8), CL02A3PW (SEQ ID NO:9) and an analogthereof.
 31. A compound comprising a peptide or peptide analog havingthe formula (IV): Z¹˜A˜M˜H˜G˜H˜H˜G˜W˜P˜W˜G˜M˜E˜Q˜G˜C˜T˜P˜L˜G_Z²  (IV)wherein: Z¹ is RRN—, RC(O)NR—, RS(O)₂NR— or an amino-terminal blockinggroup; Z² is —C(O)OR, —C(O)O—, —C(O)NRR or a carboxyl-terminal blockinggroup; each R is independently selected from the group consisting ofhydrogen, alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl,substituted arylalkyl, heteroaryl, substituted heteroaryl,heteroarylalkyl and substituted heteroarylalkyl; each “˜” independentlyrepresents an amide, a substituted amide or an isostere of an amide; andeach “-” represents a bond, or a 1 to 10 residue peptide or peptideanalog; and variants thereof in which 1, 2, 3, or 4 amino acid residuesare replaced by a different amino acid from the same class or by an Ala.32. A compound identified by the method of claim
 1. 33. A pharmaceuticalcomposition comprising a compound according to claim 25 or claim 31, anda pharmaceutically acceptable carrier, excipient or diluent.
 34. Apharmaceutical composition comprising a compound identified by themethod of claim 1 and a pharmaceutically acceptable carrier, excipientor diluent.
 35. A method of modulating IL-4 receptor-mediated IgEproduction in a cell, comprising administering to the cell an effectiveamount of a compound that binds a CLLD8 protein.
 36. The method of claim35 in which the compound competitively binds the CLLD8 protein in thepresence of an active CL02A3 compound.
 37. The method of claim 35 inwhich the compound is selected from the group consisting of CL02A3 wt(SEQ ID NO: 1), CL02A3LG (SEQ ID NO:2), CL02A3GW (SEQ ID NO:3), CL02A3TP(SEQ ID NO:4), CL02A3EQ (SEQ ID NO:5), CL02A3HG (SEQ ID NO:6), CL02A3GC(SEQ ID NO:7), CL02A3HH (SEQ ID NO:8), CL02A3PW (SEQ ID NO:9) and ananalog thereof.
 38. The method of claim 35 in which the compound isidentified by the method of claim
 1. 39. A method of treating an animalsuffering from a disease characterized by, caused by or associated withIgE production and/or accumulation and/or symptoms associated therewith,comprising administering to the animal an amount of a compound thatbinds a CLLD8 protein effective to treat the disease.
 40. The method ofclaim 39 in which the compound competitively binds the CLLD8 protein inthe presence of an active CL02A3 compound.
 41. The method of claim 39 inwhich the compound is selected from the group consisting of CL02A3 wt(SEQ ID NO:1), CL02A3LG (SEQ ID NO:2), CL02A3GW (SEQ ID NO:3), CL02A3TP(SEQ ID NO:4), CL02A3EQ (SEQ ID NO:5), CL02A3HG (SEQ ID NO:6), CL02A3GC(SEQ ID NO:7), CL02A3HH (SEQ ID NO:8), CL02A3PW (SEQ ID NO:9) and ananalog thereof.
 42. The method of claim 39 in which the compound isidentified by the method of claim
 1. 43. The method of claim 39 in whichthe disease is selected from the group consisting of an allergy, anatopic disorder, allergic rhinitis, allergic conjunctivitis, systemicmastocytosis, hyper IgE syndrome, IgE gammopathies and B-cell lymphoma.44. The method of claim 39 in which the disease is selected from thegroup consisting of an allergy, an atopic disorder, allergic rhinitis,allergic conjunctivitis, systemic mastocytosis, hyper IgE syndrome, andIgE gammopathies.
 45. The method of claim 43 in which the allergy is ananaphylactic allergic reaction.
 46. The method of claim 43 in which theatopic disorder is selected from the group consisting of atopicdermatitis, atopic eczema and atopic asthma.
 47. A kit for identifyingcompounds that modulate IL-4 induced IgE production, comprising a CLLD8protein or a cell expressing a CLLD8 protein and a compound thatcompetitively binds the CLLD8 protein in the presence of an activeCL02A3 compound.
 48. The kit of claim 47 in which the compound isselected from the group consisting of CL02A3 wt (SEQ ID NO: 1), CL02A3LG(SEQ ID NO:2), CL02A3GW (SEQ ID NO:3), CL02A3TP (SEQ ID NO:4), CL02A3EQ(SEQ ID NO:5), CL02A3HG (SEQ ID NO:6), CL02A3GC (SEQ ID NO:7), CL02A3HH(SEQ ID NO:8), CL02A3PW (SEQ ID NO:9) and an analog thereof.
 49. Amethod of inhibiting germline ε transcription in a cell, comprisingadministering to the cell an effective amount of a compound that binds aCLLD8 protein.
 50. The method of claim 49 in which the compoundcompetitively binds the CLLD8 protein in the presence of an activeCL02A3 compound.
 51. The method of claim 49 in which the compound isselected from the group consisting of CL02A3 wt (SEQ ID NO: 1), CL02A3LG(SEQ ID NO:2), CL02A3GW (SEQ ID NO:3), CL02A3TP (SEQ ID NO:4), CL02A3EQ(SEQ ID NO:5), CL02A3HG (SEQ ID NO:6), CL02A3GC (SEQ ID NO:7), CL02A3HH(SEQ ID NO:8), CL02A3PW (SEQ ID NO:9) and an analog thereof.
 52. Themethod of claim 50 in which the active CL02A3 compound is selected fromCL02A3 wt (SEQ ID NO:1), CL02A3LG (SEQ ID NO:2), CL02A3GW (SEQ ID NO:3),CL02A3TP (SEQ ID NO:4), CL02A3EQ (SEQ ID NO:5), CL02A3HG (SEQ ID NO:6),CL02A3GC (SEQ ID NO:7), CL02A3HH (SEQ ID NO:8), CL02A3PW (SEQ ID NO:9)and an analog thereof.
 53. The method of claim 50 in which the activeCL02A3 compound is selected from CL02A3 wt (SEQ ID NO:1), CL02A3LG (SEQID NO:2), CL02A3GW (SEQ ID NO:3) and an analog thereof.
 54. The methodaccording to any one of claim 1, claim 16, claim 35, claim 39 and claim49, in which the CLLD8 is a polypeptide having the amino acid sequenceof SEQ ID NO:18.